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UA / Biology / BSC 300 / What are the three main classes of membrane proteins?

What are the three main classes of membrane proteins?

What are the three main classes of membrane proteins?

Description

School: University of Alabama - Tuscaloosa
Department: Biology
Course: Cell Biology
Professor: John yoder
Term: Fall 2018
Tags: Biology
Cost: 50
Name: BSC 300-001 Exam 2 Studyguide
Description: These notes cover the chapters for our Exam 2
Uploaded: 10/05/2018
45 Pages 53 Views 10 Unlocks
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BSC 300-001 Week 5 Notes (Short because of Exam)


What are the three main classes of membrane proteins?



I. Chapter 11 – Transmembrane Transport of Ions and Small Molecules a. Overview of Transmembrane Transport

i. Movement of substances across cell membranes

1. Diffusion – spontaneous movement of material from high  

concentration to low concentration

a. The hydrophobic core of a lipid bilayer repels all but very  

small and non-polar/non-ionic molecules

b. Simple diffusion – only gases and small, non-polar  

molecules like ethanol, water and urea can freely diffuse  

across a biological membrane

i. No metabolic energy spent when moving from high  

concentration to low

ii. Rate is proportional to the molecule’s concentration  


What defines electrochemical gradient?



gradient, size, and its partition coefficient K – the  

Keq for its partition between oil and water (lipid  We also discuss several other topics like How are stars and planets contained within galaxies?

solubility)

1. Higher = more hydrophobic = faster is can  

diffuse across a bilayer

2. The first and rate-limiting step in simple  If you want to learn more check out How can language obscure meaning?

diffusion is the movement of a molecule  

from the aqueous from the aqueous  

environment into the hydrophobic interior of  

the bilayer

c. Selective permeability – all other movement across the  

membrane requires assistance of membrane associated  

proteins and is highly regulated

d. The diffusion rate of any substance across a pure  

phospholipid membrane (no proteins) is proportional to:


What is the cause of osmotic pressure?



i. Its concentration gradient across the membrane

ii. Its hydrophobicity

iii. Its size

e. Diffusion of a charged molecule is also affected by the  We also discuss several other topics like What is saltatory conduction?

membrane potential, or the separation of charge across the  

membrane

f. Electrochemical gradient – the combination of an ion/s  

concentration and the membrane potential

i. Important in establishing proton at electrochemical  

gradient to move protons

g. Three main classes of membrane proteins that transport  

molecules and ions across cellular membranes

i. Channels

ii. Transporters (uniporters, symporters, and  

antiporters)

iii. ATP-Powered pumps

BSC 300-001 Week 5 Notes (Short because of Exam)

h. Channels – protein complexes with hydrophilic interior that  provide selection of solute based on size and charge  

(usually only ions)

i. Pore = channel

ii. Most are gated channels (proteins) and are tightly  

regulated based on cell needs

1. Ligand gated

2. Ion gated – ion binds to them and alters the  

conformation to open them up

iii. Open and close in response to specific stimuli If you want to learn more check out In chemistry, what is the function of hybridization?

iv. Portion of complex blocks channel and is removed  

by conformational change following stimulus

1. Only attracted to one ion

v. Solutes move down their concentration gradient

1. Hydrophilic tube through which water  

molecules can move single file very rapidly

vi. No energy requirement

1. AKA passive – move down concentration  

gradient

2. Transporter (aka carriers) do not form channels

a. Have binding sites for specific solutes

i. Binding induces conformational change that  

promotes translocation of solute to other side of  

membrane

1. Much slower than channels

2. Hinged – opens to exoplasmic face and  

closes to open to cytoplasmic face  

depending on concentration

b. Three general categories

i. Uniporters – move a single type of molecule down  

their concentration gradients

1. Do not require energy input

2. Channels and uniporters are referred to as  

facilitated transporters

ii. Cotransporters

1. Antiporters and symporters couple the  

spontaneous transport of one solute down its  If you want to learn more check out How did kepler challenge the earth­ centered model?

concentration gradient to the unfavorable  

movement of another solute against its  

concentration gradient

a. Conformation change driven by the  

spontaneous process allows the non

spontaneous process to occur

i. Allows movement of one  

substance down the  

concentration gradient,

BSC 300-001 Week 5 Notes (Short because of Exam)

altering the conformation of  

transporter to allow another  

to move against its  

concentration gradient

b. Symporter – moving in same  

direction

c. Antiporter – moving in opposite  

directions

ii. Active transport requires ATP hydrolysis

1. ATP-Powered pumps use hydrolysis of ATP to drive conformation  change in these unidirectional transporters

a. Transporters move solutes against concentration gradients  (usually alters conformation)

i. This is active transport

iii. Collective activity of multiple types of transporters needed for many  physiological functions We also discuss several other topics like When are polygraph tests illegal to use?

1. Cell utilizes them to reserve potential energy to move antiporters  or symporters

2. Lysine uptake across plasma membrane requires three transporters: a. Na+/K+ pump – ATP powered pumps that moves Na+ out  of and K+ into the cell

b. K+ channel – passive transporter – K+ diffuses down  

concentration gradient, establishing a powerful  

electrochemical gradient across the membrane (membrane  

potential – high positive concentration outside of the cell)

c. The charge across the membrane gives Na+ a lot of  

potential energy

i. Diffuses down its electrochemical gradient through  

a symporter that moves lysine against its  

concentration gradient

iv. Uniport transport is faster and more specific than simple diffusion 1. Distinguishing uniport from simple diffusion

a. Rate of movement by uniporters is much higher than  

simple diffusion

b. Partition coefficient of solute is irrelevant because the  

molecule never interacts with the hydrophobic core of the  

bilayer

c. The maximum transport rate (Vmax) depends on the  

number (concentration) of uniporters

d. Transport is reversible

i. If the gradient changes, so will the direction of  

transport

e. Specificity – each transporter has high specificity for a  

single molecule or ion or group of very similar ones

i. As in enzyme kinetics, Km is the substrate  

concentration at ½ Vmax

BSC 300-001 Week 5 Notes (Short because of Exam)

2. One of the best understood uniporters is the glucose transporter  GLUT1, found in the plasma membrane of most mammalian cells b. Facilitated Transport of Glucose and Water

i. Human GLUT1 uniporter facilitates the glucose transport into cells 1. Uniporters randomly change conformation (flipping back and forth  between open to inside and outside), exposing solute-binding site  to one side of the membrane then the other

2. Binding of the solute does not change the conformation at all,  which is what allows it to flip back and forth

3. Transition occurs through an intermediate state where the solute is  inaccessible – occluded (no solute binding state)

4. When the transporter is saturated, the rate of transport is maximal a. The rate, Vmax, is characteristic for each transporter

b. Each transporter has a specific affinity for its solute Km  

that is equal to the concentration of solute when the  

transport rate is ½ of its max value

5. The low Km of GLUT1 allows it to transport glucose into most  mammal cells

a. Glucose concentration is higher in extracellular medium  

than inside cells – average of 5mM blood glucose levels

b. GLUT1 (red blood cells) Km is 1.5mM

c. GLUT2 (liver cells) Km is 20mM

i. At this concentration those cells are taking up very  

small amounts of glucose – glucose in those cells  

are used for a different purpose (long-term sugar  

storage)

d. Lower Km = greater affinity = more rapid transport

e. Cellular concentration of glucose is kept low by it being  

immediately converted to glucose-6-phosphate by the first  

enzyme of glycolysis

i. This promotes constant uptake of glucose by RBCs

ii. Simple enzyme kinetics of glucose mediated uptake by GLUT transporters 1. At least 14 distinct GLUT transporters encoded by the human  genome

a. Most body parts are in GLU1

b. Each is expressed in different subsets of cells, and each  

with a distinct Km for glucose, allowing distinct responses  

to changes in blood glucose levels

i. Ex: GLUT2 is expressed in the liver and pancreatic  

β cells

c. Following a meal blood glucose levels almost double  

(10mM)

i. Barely affects the import of glucose into RBC’s due  

to GLUT 1’s low Km

ii. But in liver and β cells, glucose uptake it doubled

BSC 300-001 Week 5 Notes (Short because of Exam)

1. This allows its conversion to glycogen in the  

liver and β cells to be stimulated to release  

insulin

a. Liver cells polymerize glucose to  

glycogen – release insulin and tell  

cells to take up glucose in the  

process

iii. Osmotic pressure causes water to move across membranes 1. Osmosis - movement of water from low solute concentration to  high solute concentration to reach equilibrium

2. Proper water balance in cells is critical for physiology, shape,  rigidity (turgor pressure)

3. Osmotic pressure – hydrostatic pressure required to stop the net  inward flow of water

4. Hypotonic solution – cells swell

a. Concentration of non-membrane penetrating solutes is  

lower than in the cytosol

b. Osmotic pressure may not be able to overcome influx in  hypotonic solution, so cells must regulate ionic  

concentrations to prevent rupture

5. Hypertonic solution – cells shrink

a. Concentration of non-membrane penetrating solutes is  

higher than in the cytosol

6. Isotonic solution – cells remain unchanged

iv. Aquaporins increase water permeability of cell membranes 1. Movement of water into some cells is also critical for their  function

a. Ex: kidney cells that reabsorb water from urine

2. Aquaporins – specialized channels that allow passive movement of  water

a. Homotetramer generates a small channel just wider than a  water molecule

i. Lined with hydrophilic residues that allow water  

molecules to move single file down their osmotic  

gradient into the cell

1. From one side of the membrane to another

2. Can move protons

ii. Specificity is high enough to even select against  

protons associated with water molecules

1. Maintains appropriate osmotic pressure in  

many cell types

2. In kidney epithelial cells they promote water  

reabsorption from urine – mutations  

associated with diabetes insipidus – very  

dilute urine

a. Allows right proportions of urine

BSC 300-001 Week 5 Notes (Short because of Exam)

c. ATP-Powered Pumps and Intracellular Ionic Environment

i. Important Concepts:

1. Four classes of transmembrane proteins couple energy released by  ATP hydrolysis with energy-requiring transport of substances  

against their concentration gradients

2. Combined action of P-class ATP-powered pumps generates the  steady state ionic balance of animal cells

3. ABC superfamily proteins transport a wide array of substrates,  including toxins, drugs, phospholipids, peptides, and proteins, into  or out of the cell

ii. Four classes of ATP-powered transport proteins

1. P-class

a. Moves only ions

b. Terminal phosphate group of ATP is transiently covalently  

bound to transporter

c. Alters conformation and drives movement of ions against  

electrochemical gradients

i. Large protein family

ii. All are heterotetramers

d. Ex: Sodium-Potassium pump

2. V-class “Vacuolar-type H+ ATPase”

a. Moves only ions

b. More complex transporter type

i. Multiple transmembrane and peripheral proteins

ii. No phosphoprotein intermediate

iii. Protons are the only solutes transported

1. Out of cytosol and into vacuoles of yeast,  

fungi, and plants

2. Into lysosomes and endosomes of animal  

cells

3. Across plasma membrane of osteoclasts and  

some kidney tubule cells

3. F-class

a. Moves only ions

b. Structurally and functionally similar to V-class pumps

i. No phosphoprotein intermediate

c. Likewise no phosphoprotein intermediate

d. These are reverse proton pumps – allow protons to flow  

down electrochemical gradient

i. Allow protons to flow rather than moving them

ii. High concentration to low

e. Energy released turns pump and associated conformational  

changes drive ATP synthesis in mitochondria and bacteria

f. AKA ATP synthases

4. ABC (ATP-Binding Cassette) superfamily

a. Moves a wider variety of solutes

BSC 300-001 Week 5 Notes (Short because of Exam)

b. No phosphoprotein intermediate

c. Large superfamily with shared core structure (don’t worry  about the nature of these below)

i. 2 transmembrane domains

ii. 2 cytosolic ATPase domains

iii. Couple ATP hydrolysis to solute movement

iv. Some are importers, others exporters

d. Several hundred superfamily members, each transporter  specific for single substrate or small group: ions, sugars,  

amino acids, phospholipids, cholesterol, peptides,  

polysaccharides, proteins and various toxins/drugs

e. Some are inappropriately expressed in certain cancer cells  allowing efficient export of anti-cancer drugs resulting in  

multi-drug resistance

i. Move lipiphilic drugs out of cells when they are  

expressed in cancer – makes cancer resistant to  

chemo

f. One of the most ancient transporters

iii. Sodium/Potassium P-type ATPase Pump

1. Tetramer – 2 α (catalytic) and 2 β (structural) subunits

2. Transporter phosphorylated and dephosphorylated during cycle 3. Different phosphorylation and solute binding states promote  conformational change and solute affinity that promote  

translocation of Na+ and K+ against electrochemical gradients 4. Transports 3 Na+ out and 2 K+ into cell

5. Ions are pumped in both directions across the membrane – each ion  moves against its concentration gradient

6. E1 conformation

a. 3 high affinity Na+ binding sites and two low affinity K+  binding sites accessible from cytosolic surface of protein

b. Km for Na+ binding to cytosolic sites is 0.6 mM - much  lower than the intracellular Na+ concentration of about 12  

mM

i. Na+ ions fully occupy sites

ii. High affinity for 3 Na ions here

c. Affinity for cytosolic K+ binding sites is so low that K+  ions, transported inward through the protein, dissociate  

from E1 and enter the cytosol despite the high intracellular  

K+ concentration

7. E1 to E2 transition:

a. 3 Na+ ions bind high affinity sites and ATP binds a high  affinity site

i. Alters conformation and pump closes

b. New conformation activates ATPase activity, causing  

pump to hydrolyze ATP and phosphorylate itself on a  

conserved Aspartate residue

BSC 300-001 Week 5 Notes (Short because of Exam)

i. Alters conformation and pump opens to exoplasmic  

face

1. Binding affinities have changed

c. Eventually goes back to E1 conformation

d. Do not have to memorize the exact steps and their numbers 8. E2 conformation

a. Low affinity for Na+

i. Ions released, even though extracellular Na+  

concentration is high

b. Conformation has high affinity for two K+ which are  

bound

i. Alters conformation to a closed state and induces  

release of Pi

1. Opens pump to original cytoplasmic  

conformation

2. 2 K+ are released

9. Overall

a. Cells use 25% to 50% of all available ATP to drive the  

Na+/K+ ATPase

b. The electrochemical gradient it produces is a powerful  

energy source for importing and exporting metabolites and  

conducting neural impulses

iv. Operational model of Ca2+ P-type ATPase

1. Very similar to Na+/K+ pumps, but single ion species and only in  one direction

a. One ion in one direction instead of 2 in different directions 2. Pump establishes stores of Ca2+ in the smooth ER

3. Regulated release of Ca2+ controls activity of many cytoplasmic  proteins – especially important in Ca2+ dependent muscle  

contraction

4. Calmodulin, a cytosolic Ca2+ binding protein, regulates activity of  plasma membrane Ca2+ ATPases

v. Acidifying organelle requires moving more than protons 1. V-class proton pumps acidify organelles like lysosomes, but  require help from other transporters

2. Acting alone would lead to negative charge build-up in cytoplasm 3. Protons and cytoplasmic ions would be attracted across the  organelle membrane (electric potential) preventing diffusion of  protons and impeding import of additional protons – no change in  pH

4. Therefore, acidification of organelles, like lysosomes, requires  addition inward pumping of Cl- ions to minimize this electric  potential

a. Pumps Cl- into lumen

b. Freely diffusing inside lumen of lysosome

vi. ABC transporters and resistance to cancer drugs

BSC 300-001 Week 5 Notes (Short because of Exam)

1. Over 50 mammalian ABC transporters

a. Many with specificity for molecular toxins, including  medically important drugs

2. ATP hydrolysis used to pump these drugs into extracellular space 3. Many cancer tumor cells acquire mutations that overexpress  certain ABC transporters (specifically ABCB1), making them  highly efficient at exporting membrane soluble drugs

4. Such multidrug resistant cells are much more difficult to treat with  traditional chemo

5. Flippases are ABC transporters

6. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) a. Peculiar ABC transporter

i. No longer functions in active transport, but instead  

is a passive ATP-regulated chloride ion channel

1. No longer pumps – it is now just a  

channel for Cl- to move into extracellular  

space

ii. Allows Cl- ions to diffuse out of epithelial cells in  

the lungs, liver, pancreas, digestive tract,  

reproductive tract, and is necessary for reuptake of  

Cl- following sweating

iii. In the lungs activated CFTR secretes Cl- into the  

respiratory tract which draws water out of the cells

iv. Water lowers viscosity of secreted mucus allowing  

infectious bacteria to be cleared

b. Cystic fibrosis

i. Disease where breakdown in number of activities in  

different tissues

ii. Most die of complications of respiratory systems

iii. In the absence of CFTR protein, decreased Cl- ions  

and increased H2O absorption leads to dehydration  

of the mucus layer

iv. Pathogenic bacteria cannot be cleared

v. Most result from 3 base pair deletion (ΔF508  

mutation – eliminates a single amino acid) that  

precents protein from folding correctly at normal  

body temp – therefore never reaches cell membrane

1. Most proteins are destroyed in the ER  

because of improper folding

2. Can fold properly at low temp but not body  

temp

vi. Clinical trials using liposomes to deliver normal  

copies of the gene are under way

vii. First report (2015, Lancet) of 136 patients receiving  a yearlong regimen of inhaled liposome-CFTR

BSC 300-001 Week 5 Notes (Short because of Exam)

showed modest, but significant improvement in  

lung function

1. Liposomes as delivery for drugs

viii. Other treatments include small molecules that bind  

the nascent protein and help it fold correctly

d. Nongated Ion Channels and the Resting Membrane Potential

i. Important concepts:

1. Ionic concentration gradients across animal cell membranes  

establishes a resting potential – similar to separation of electrical  charge within a battery

2. Magnitude is approximately -70mV with the cytoplasmic face of  the PM negatively charged with respect to the exoplasmic face

3. Animal cell plasma membrane resting potential is generated by the  ATP-powered Na+/K+ pump and non-gated resting K+ channels

4. This membrane potential is used to drive a number of cells,  

including neural impulses

ii. Nerve cells communicate through electric impulses

1. Nerve cells collect and conduct info as fast-moving electrical  impulses

2. Dendrites – receive info, usually from other nerves

a. Make synapses with other neurons to receive signals

3. Axons – transmit info to other cells via neural impulses and release  of neurotransmitters

a. Cell body stimulates – depolarization sweeps along axon  

where it has synapsed with another cell – secretes  

neurotransmitter

b. Axons of vertebrates are insulated by myelin sheaths  

composed of Schwann cells which produce a thick lipid  

rich membrane (myelin)

i. Schwann cells rich in sphingolipids

ii. Wrap around axon and prevent negative charge  

inside from interacting with positive charge outside

iii. Speeds up neural processes

e. Voltage-gated ion channels and the propagation of action potentials i. Establishing resting potential

1. In the resting state there is only one type of active ion channel – resting K+ channels

a. AKA potassium leak channel – always open

i. Leaves behind a lot of negative charge on the  

cytosolic side of the face of the plasma membrane  

that establishes membrane potential

2. While concentration differences across the membrane favor the  exit of K+ from the cell, the growing negative charge inside favors  its retention

3. When opposing forces are balanced no further net movement of  K+ occurs

BSC 300-001 Week 5 Notes (Short because of Exam)

a. K exiting leaves behind a negative charge to attract K cells  that stop leaving the cell, establishing equilibrium

4. This establishes the resting potential and makes movement of K+  out of the cell the most important factor in determining resting  potential

5. Resting potential is calculated using the Nerst equation, which  depends on the concentration difference between K+ inside of and  outside of the cell

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a. Decrease in positive charge in the cytosol results in an  

electric potential across the membrane proportional to that  

difference

i. Weak permeability of membrane to Na+ ions raises  

potential to -70 mV

ii. The Action Potential

1. Excitation and neural impulses involve opening/closing of voltage  gated Na+ and K+ channels

2. When a nerve cell is stimulated a small number of Na+ channels  open

a. Na+ diffuses back into cell body

b. Membrane potential rises

3. If enough Na+ enter, the membrane potential reaches a threshold  of approximately -50mV

4. All voltage-gated Na+ channels in cell body then open and the cell  becomes depolarized: rapid jump to approximately +40mV

a. Depolarization – rapid Na+ influx creates positive

membrane potential, reaching equilibrium potential of Na+,  about +40mV

b. In a fraction of a second these channels are shut off by  

channel inactivating segments – tethered domains that  

block the channel an lock it in using a conformation that  

cannot be stimulated

i. Makes them refractory to being re-stimulated

c. Increased Na+ concentration activates voltage-gated K+  channels and K+ rapidly exits the cell

d. Along with the ever active Na+/K+ pumps membrane  

returns to resting potential

e. Collectively, these changes in membrane potential  

following excitation are referred to as action potential

5. Initiation of an action potential is an all or nothing event a. Sub-threshold stimulation will elicit no action potential

b. Stimulation at or above the threshold stimulates the same  degree of action potential

BSC 300-001 Week 5 Notes (Short because of Exam)

c. Stronger stimuli (scalding water) “feel” stronger because  more neurons are activated or “high-threshold” neurons are  activated that would otherwise remain at rest

6. Energy independent process, depending online on the diffusion of  ions down their concentration gradients

7. Local anesthetics, like Novocain, act by closing ion channels and  preventing action potentials

8. Propagation of Action Potential and the nerve impulse a. Following depolarization, the localized influx of Na+  stimulates opening of Na+ channels adjacent to the site of  depolarization

b. Propagation only goes in one direction due to the refractory  period that follows depolarization

c. Neural impulse speed

i. Two principle mechanisms have evolved to speed  

up neural impulses:

1. Nerve diameter – wider neurons have less  

reistance to current flow (Na+ ions can  

move faster)

a. Many invertebrates, like mollusks,  

have evolved very wide neurons

2. Insulation – vertebrate specific Schwann  

cells and oligodendrocytes wrap axons in a  

myelin (lipid) sheath

a. Increases rate of conductance by  

removing the interference of anions  

at the membrane

ii. Na+ ions rapidly travel toward axon terminus, but  

signal strength decreases

iii. Nodes of Ranvier – spaces between myelin sheaths  

where all Na+ activated Na+ channels are located

1. When the otential reaches next Node of  

Ranvier, rapid influx of Na+ ions amplifies  

the potential again

2. This step-wise conductance moves impulses  

at approximately 120 m/s

a. Almost 20 times faster than in un

myelinated axons of similar diameter

b. Called saltatory conduction

iv. Disease implication

1. Multiple sclerosis – an autoimmune disease  

characterized by damage to the myelin  

sheath leading to decreased neuronal  

conductance

2. Varied symptoms:

a. Visual disturbances

BSC 300-001 Week 5 Notes (Short because of Exam)

b. Muscle weakness

c. Trouble with coordination and  

balance

d. Sensations such as numbness,  

prickling, or “pins and needles”

e. Thinking and memory problems

BSC 300-001 Week 6 Notes

I. Cellular Energetics Part I

a. First Step of Harvesting Energy from Glucose: Glycolysis

i. Important Concepts

1. As electrons move from being evenly shared to not, they give  

energy up to the system

a. Cells cannot take glucose blowing up, so it is done in steps,  

and the energy is coupled to less favorable reactions

2. Aerobic oxidation – cells use a four-stage process to transfer  

energy released by glucose/fatty acid oxidation into the terminal  

phosphoanhydride bond ATP

3. Glycolysis – Stage 1: Cytosolic enzymes partially oxidize glucose  of 2 molecules of pyruvate and generate two molecules each of  

NADH and ATP

a. Glycolysis does not require oxygen and is an anaerobic  

process

b. In the cytoplasm of eukaryotic cells

c. Represents partial oxidation of glucose

d. Some energy trapped in ATP bonds

e. Rest of the energy is captured in electrons in NADH

4. Fermentation – in the absence of oxygen some cells can reduce  

pyruvate to lactic acid or ethanol and CO2 in order to oxidize  

NADH back to NAD+ required for glycolysis

a. Most cells will die if Oxygen is not present

i. A few can use pyruvate to resupply cells with NAD  

electron acceptor to maintain small production of  

ATP

ii. Metabolism

1. The collection of biochemical reaction that occur within a cell, or  

the net sum total of reaction taking place in our cells

2. Metabolic pathways – sequential chemical reactions linked by  

enzyme catalyzed steps

a. Each reaction in the sequence is catalyzed by a specific  

enzyme that produces metabolic intermediates which are  

substrate for downstream enzymes and ultimately leads to  

the formation of a final product

b. Interconnected at various points so that a compound  

generated by one pathway may be shuttled in a number of  

directions, depending on cellular requirements

c. Metabolic pathways can be catabolic or anabolic (likely be  

a test question)

i. Catabolic pathways – break down complex  

substrates into simple end products

1. Provides raw materials for the cell

2. Transfers chemical energy from complex  

molecules to the high energy bonds of  

NADH and ATP

BSC 300-001 Week 6 Notes

a. Releases very low free energy  

molecules such as CO2 and H2O

ii. Anabolic pathways – synthesize complex end  

products from simple substrates

1. Requires energy

2. Uses ATP and NADPH from catabolic  

pathways

iii. Overview of aerobic oxidation and photosynthesis

1. Two principle eukaryotic processes convert external energy into  ATP – aerobic respiration and photosynthesis

2. Aerobic respiration – reduced organic molecules (sugars, lipids)  are oxidized to generate high energy electron carriers (NADH and  FADH2) which power oxidative phosphorylation – the synthesis of  ATP from ADP and Pi

a. Proton-motive force established – protons sequestered to  one side of a membrane system and their electrochemical  

gradient drives ATP synthesis by powering F-type ATP  

pumps

i. High-energy electrons generated by:

1. Light absorption by pigments (eg  

chlorophyll)

2. Catabolism of sugars and lipids and carried  

in the reduced form of electron carriers (eg  

NADH and FADH2)

ii. Electrons release energy as they pass through an  

electron-transport chain

iii. Released energy is used to pump protons across the  

membrane, generating the proton-motive force

iv. Collectively called chemiosmosis

1. Chemiosmotic coupling – proton motive  

force releases energy as protons flow down  

electrochemical gradient

a. Drives synthesis of ATP

b. And other processes like transport of  

metabolites across the membrane  

against their concentration gradient  

as well as rotation of bacterial  

flagella

3. Photosynthesis – cells use energy of photons to excite electrons  into high energy states

a. Electrons also reduce electron carriers which drive the  

synthesis of ATP and sugar

b. Proton-motive force also established

iv. Cellular Respiration

1. Cellular metabolic reactions that convert biochemical energy from  nutrients into ATP and release the waste products CO2 and H2O

BSC 300-001 Week 6 Notes

a. Catabolic reactions that break large molecules into smaller  ones, releasing energy as less stable high-energy bonds are  replaced by more stable (lower free energy) bonds

b. Considered an exothermic oxidation-reduction (redox)  reaction

i. Redox reaction composed of two half reactions:  

oxidation and reduction

1. Molecule being oxidized donates electrons  

to the molecule being reduced (aka electron  

acceptor)

a. May be total transfer – ionization

b. May be partial transfer – new polar  

covalent bond formation

2. Electrons move toward the more  

electronegative atom

a. Gives up energy when they move  

closer to oxidizing agents

b. Energy trapped in catalytic sites to  

be used to catalyze endergonic  

reactions

3. When a substrate gains electrons, it is  

reduced

4. When a substrate loses electrons, it is  

oxidized

5. More hydrogen – more reduced – more  

stored energy

6. More oxygen – more oxidized

2. Metabolism

a. Oxidation of sugar – cellular respiration

i. Glycolysis – first stage in the oxidation of glucose 1. Partially oxidizes glucose in the soluble  

portion of the cytoplasm of eukaryotic cells

ii. Citric Acid Cycle – completes oxidation of glucose 1. Second stage of cellular respiration and  

occurs in the mitochondria of eukaryotic  

cells (aka Tricarboxylic Acid Cycle or  

Kreb’s Cycle)

iii. Very few ATP are generated during glucose  

oxidation

1. Instead, the energy released by its oxidation  

reduces “high-energy” electron carriers  

(primarily NAD+) that deliver electrons to  

the mitochondria

a. Therefore, ETC converts the energy  

of these electrons to ATP

b. Complete oxidation of glucose

BSC 300-001 Week 6 Notes

!"#$%&" + 6&% → 6!&% + 6#%&

i. Net ∆+°- = −686 2345/785

ii. Most of this energy is released as heat (entropy of  surroundings increase)

iii. Remaining energy is captured in newly formed  terminal phosphoanhydride bond of ATP

c. ATP and ATP Hydrolysis

i. Terminal phosphate bond is an unstable high-energy  bond that can easily be broken in the presence of  

water or other nucleophiles

1. On 5’ Carbon

ii. The release of free energy results from the  

formation of more stable products ADP and  

inorganic phosphate (Pi)

1. In cells, ATP is rarely hydrolyzed directly to  

ADP and Pi – exception: muscle cells in  

response to low temps

a. Such hydrolysis releases all free  

energy as heat and produces the  

shivering effect we experience on  

cold days

2. In cells, ATP hydrolysis is a multistep  

process catalyzed by enzymes or a series of  

enzymes

a. Hydrolysis takes place as the  

phosphate group is removed from  

ATP and transferred to a new  

substrate molecule

b. Such phosphorylation raises the free  

energy of the molecule receiving the  

phosphate group

i. Allows new phosphor

molecule to undergo a  

subsequent spontaneous  

reaction that involved the  

transfer of the phosphate  

group to another molecule, or  

in the terminal reaction, to be  

released as Pi

c. ATP hydrolysis is complete when a  

free inorganic phosphate is released  

into the solution

i. The multiple resonance  

structures of inorganic  

phosphate make it a very

BSC 300-001 Week 6 Notes

stable – low energy –

molecule

d. Before we can make any ATP,  

however, we have to extract the  

energy of glucose (must be oxidized)

i. Two enzymatic steps in  

glycolysis catalyze the  

oxidation of glucose  

metabolites and the reduction  

of NAD+ to NADH  

(Nicotinamide adenine  

dinucleotide)

ii. Reduction of co-enzyme  

NAD+ is an endergonic, non

spontaneous process that  

requires it to be coupled to a  

favorable, exergonic process

iii. When NAD+ is reduced it  

accepts two electrons and one  

hydrogen from the enzyme  

substrate

v. Glycolysis: Partial Oxidation of Glucose

1. General

a. Occurs in the cytoplasm

b. Glucose to pyruvate in 10 enzymatically catalyzed  

reactions

i. Steps 2,4,5,6,8, and 9 are endergonic and non

spontaneous (positive G)

!"#$%&" + 2:;< + 2<= + 2>:;? → 2 @ABCD4EF + 2:G< + 2>:;# + 2#? + 2#%& ii. In cells, the ΔG for all reactions is favorable

1. For all but 3 of these reactions the ΔG is  

almost 0 (near equilibrium)

a. These 3 reactions (1,3 and 10) have  

very large negative ΔG values,  

making them irreversible under  

normal conditions

i. They drive the entire process  

in the forward direction

ii. They are major points of  

regulatory control of  

glycolysis (allosteric  

regulation)

2. Steps

a. We need to know names of enzyme substrate and product  

for steps 1,3,6/7, and 10

BSC 300-001 Week 6 Notes

i. Step 1 – activate glucose with addition of phosphate  1. Glucose phosphorylated to glucose 6-

phosphate

2. Enzyme – hexokinase  

3. Allosteric regulation – the product glucose

6-phosphate is a negative allosteric regulator

of hexokinase

a. product is cut off (hexokinase is  

inhibited by glucose-6-phosphate)

4. Glucose 6 phosphate allows it to import  

more glucose

ii. Step 2 – Glucose 6-phosphate is isomerized to  fructose 6-phosphate

iii. Step 3 – Fructose 6-phosphate is phosphorylated to  fructose 1,6- biphosphate using another ATP

(NEED TO KNOW THIS STEP WELL)

1. Second irreversible step

a. Also the committed step –

irreversible step for which the  

product will be used solely for the  

completion of that pathway

2. Steps 2-3 essentially rearrange the molecule  to create near perfect symmetry

3. Phosphofructokinase-1 is the most important  control enzyme in the glycolytic pathway

a. Allosterically inhibited by ATP and  

citrate

b. Allosterically activated by AM and  

F2, 6pB – production regulated by  

hormones insulin and glucagon

4. Fructose 1,6-biphosphate (F1,6bP) is an  

allosteric activator of pyruvate kinase, the  

last enzyme of the glycolytic pathway

iv. Step 4 – F1, 6bP is split into the 3 carbon molecules  glyceraldehde 3-phosphate and dihydroxyacetone  phosphate

1. Cleavage

2. Made from symmetrical F1,6bP

v. Step 5 – an isomerase interconverts the two  molecules and Glyceradehyde-3-phosphate (G3P) is  siphoned off by step 6

1. Only metabolite that is used by the next  

enzyme

vi. Steps 6-10 – energy is extracted from G3P and its  metabolites in the form of 2 NADH and 4 ATP  molecules

BSC 300-001 Week 6 Notes

1. Step 6 – oxidation of G3P to 1,3-

biphosphoglycerate (catalyzed by  

glyceraldehyde 3 phosphate dehydrogenase)  

is energetically favorable and is coupled to  

reduction of NAD+ to NADH and  

phosphorylation of G3P

a. Dehydrogenase enzymes oxidize and  

reduce cofactors or coenzymes

i. Remove Hydrogen (1 proton  

and 2 electrons) and pass it  

onto coenzyme NADH

b. NAD+ is a glyceraldehyde  

phosphate dehydrogenase coenzyme  

that is reduced in this reaction

2. Step 7 – 1,3-Biphosphoglycerate has  

sufficient transfer potential to donate its  

phosphate group to ADP

a. Enzyme phosphoglycerate kinase  

catalyzes this substrate level  

phosphorylation: the transfer of a  

phosphate group from an enzyme’s  

substrate to ADP

i. ADP to ATP

ii. We have paid back the ATP  

debt

3. Steps 8-9 – 3-phosphoglycerate is converted  to phosphoenolpyruvate, a phosphorylated  

molecule with higher transfer potential than  

ATP

a. Basically make a molecule with  

higher electron transfer potential by  

rearranging bonds to make  

phosphopenolpyruvate

4. Step 10 – final irreversible step

a. Pyruvate kinase catalyzes the  

transfer of phosphate group from  

PEP to ADP, generating 2 additional  

molecules of ATP per glucose  

molecule via substrate level  

phosphorylation

b. Final product of glycolysis, pyruvate,  

is generated

c. Allosteric regulation –

phosphofructokinase is negatively  

regulated by ATP (glycolysis slows  

down when more ATP is present)

BSC 300-001 Week 6 Notes

vii. Big things to know:

1. Importance of 3 irreversible steps

2. Review what dehydrogenase enzyme does in  

step 6 – important for Thursday citric acid  

cycle lecture

3. End of Glycolysis

a. Pyruvate molecules are still highly reduced – only partially  

oxidized – still energy left

b. Glycolysis generates a net of 2ATP’s for each glucose

i. 4 ATP produced but 2 are consumed

ii. 2 ATP + 2ADP yields 4 ATP

c. Other sugars besides glucose can enter the pathway by  

forming one of several sugar intermediates

d. Anaerobic process

e. Pyruvate (end product) can enter aerobic or anaerobic  

catabolic pathways

vi. Anaerobic Oxidation of Pyruvate – The Fermentation Process 1. Fermentation restores NAD+ from NADH by reducing pyruvate a. Under anaerobic conditions, glycolysis is the only source of  

ATP

i. Glycolysis depletes the supply of NAD+ by  

reducing it to NADH – glycolysis cannot continue  

without a supply of NAD+

2. In muscle and tumor cells pyruvate is reduced to lactate

3. In yeast and other microbes, pyruvate is reduced and converted to  ethanol

4. Fermentation is inefficient with only 8% of the energy of glucose  captured as ATP

b. The Structure and Functions of Mitochondria

i. Bean-shaped organelles but may be round or threadlike

ii. Size and number of mitochondria reflect the energy requirements of the  cell

iii. Many functions – do not need to know specifics

iv. Outer mitochondrial membrane

1. Most abundant protein is the porin VDAC (voltage dependent  anion channel)

a. Permeable to ions and small proteins

v. Inner Mitochondrial membrane

1. Major permeability barrier to the mitochondrial matrix

2. Has two interconnected domains:

a. Boundary membrane – adjacent to outer membrane – held  

in contact by protein complexes called MICO’s

b. Cristae – interior folds where the protein complexes  

involved in ATP synthesis are locate

i. These complexes are segregated from boundary  

membrane by MICO’s

BSC 300-001 Week 6 Notes

3. Mitochondrial matric interior of inner membrane

a. Contains multiple copies of the circular mt chromosome,  ribosomes, and diverse enzymes

vi. Intermembrane space

1. Between two membranes into which protons are pumped to charge  the electron transport system

vii. In mammals 99.99% of mitochondria is maternally inherited (sperm  mitochondria rarely enter egg)

viii. Evolutionarily derived from bacterial ancestor, but highly reduced genome  – degree of reduction varies among organisms, but very small number of  genes

1. Vast majority of genes required for mt functions are nuclear 2. The few genes left in the mt genome all encode components of  electron transport system, as well as tRNAs and mt ribosomal  RNA

a. None exported to cytoplasm

3. Like bacteria, inner mt membrane lacks cholesterol and is rich in  the lipid cardiolipin

4. Also similar to bacteria, a few codons are non-standard

5. Sequenced mitochondrial genomes suggest they are derived from a  member of the Rickettsiaceae bacteria group, a family of obligate  intracellular parasites

ix. Mutation in mitochondrial DNA linked to several human diseases 1. Because mitochondria are present in multiple copies, with each  being copied and inherited separately, genetic mitochondria  

mutation exhibits heteroplasmy

a. Mixed genotype within and between mitochondria of the  same organism

2. Segregate randomly into daughter cells, which can lead to  heterogeneous cell populations with poor mitochondrial function 3. Accumulation of mutation contributes to general age-related  defects in cell physiology

a. High rate of metabolism of mitochondria elevates mutation  rate through production of reactive oxygen species

b. Mutation of specific genes associated with some human  

diseases such as optic neuropathy, progressive  

ophthalmoplegia, and muscle defects

x. Mitochondria can rapidly fuse with one another, or split in two 1. Fusion of mitochondria can help offset accumulation of deleterious  mutations by sharing “good” copies of genome

2. Cells can recognize poorly functioning mitochondria and promote  mitophagy – subset of autophagy – in which ER derived  

membranes surround the poorly functioning organelle then fuse  with lysosomes, which degrade the mitochondria

xi. Mitochondria influenced by direct contact with ER

BSC 300-001 Week 6 Notes

1. ER mitochondria associated membranes (MAMs) regulate mt  

fusion and fission

2. Calcium influx from MAMs to mitochondria stimulate ATP  

synthesis (via Ca2+ dependent function of citric acid cycle  

enzymes)

3. Also helps activate signals from mitochondria that promote  

apoptosis (Ca2+ overload in mitochondria leads to activation of  

apoptosis)  

II. Cellular Energetics Part 2

a. The Citric Acid Cycle and Fatty Acid Oxidation

i. Important Concepts

1. The mitochondrial transmembrane complex pyruvate  

decarboxylase links glycolysis with the Citric Acid Cycle by  

oxidizing pyruvate, reducing NADH, and releasing acetyl-CoA  

into the matrix

2. Acetyl-CoA is further oxidized to 2 molecules of CO2 in the Citric  Acid Cycle – releasing additional electron carriers NADH and  

FADH2

3. Therefore, most of the energy released in oxidation stages I and II  

is temporarily stored in reduced NADH and FADH2, which carry  

high-energy electrons that subsequently drive electron-transport  

chain (Stage III)

4. Oxidation of short-to long-chain fatty acid occurs in mitochondria  with production of ATP and additional NADH

ii. Introduction

1. Early Earth populated by anaerobes, organisms that capture and  

utilize energy by oxygen-independent metabolism

2. Oxygen accumulated in the primitive atmosphere after  

cyanobacteria appeared and began to photosynthesize, increasing  

oxygen levels in the atmosphere

a. The presence of oxygen drives the reaction for energy

3. Aerobes evolved to use oxygen in order to extract more energy  

from organic molecules

a. In eukaryotes aerobic respiration takes place in the  

mitochondria

iii. Summary of aerobic oxidation of glucose and fatty acids

1. Stage 1 – cytosol

a. Glucose partially oxidized to pyruvate

b. Fatty acid. Esterified to CoA

c. Pyruvate and fatty acid transported through inner  

membrane into matrix

d. Cytosolic NADH electrons shuttled to NAD+ in the matrix

2. Stage 2 – Mitochondrial matrix: Pyruvate Dehydrogenase, Citric  

Acid Cycle, and Fatty Acid Oxidation

a. Pyruvate oxidized to acetyl CoA and CO2 with reduction  

of NADH

BSC 300-001 Week 6 Notes

b. Fatty acyl CoA oxidized to release acetyl CoA and  

additional FADH2/NADH

c. Acetyl CoA oxidation in the Citric Acid Cycle generates  NADH and FADH2, GTP, and CO2

3. Stage 3 – Inner Membrane: Electron Transport and Proton Motive  Force

a. NADH and FADH2 transfer energetic electrons to electron  transport chain

b. Electrons pass through ETC complexes to ultimate  

oxidizing agent O2

c. Energy released by their diffusion drives H+ transport from  matrix to intermembrane space

i. Generates the electrochemical gradient comprising  

the proton-motive force

4. Stage 4 – Inner membrane and matrix: Oxidative Phosphorylation a. ATP synthase harnesses proton-motive force energy to  

generate ATP in the matrix

b. Antiporter proteins – import ADP and Pi into the matrix  coupled to export of ATP and hydroxide ions

iv. Oxidative Metabolism in the Mitochondrion

1. Pyruvate Dehydrogenase Complex links Glycolysis to TCA Cycle a. Pyruvate dehydrogenase – high protein complex that  

catalyzes this critical 3-step reaction

i. Critical coenzymes are NAD+ and Coenzyme A

ii. Pumped into matrix by proton symporter

iii. Step 1 – oxidizes pyruvate, releasing CO2

1. Exergonic – coupled to

2. Acylation of Coenzyme A forming acetyl  

CoA

3. Reduction of NAD+ to NADH

iv. Major site of allosteric regulation:

1. Positive – fructose-1,6 biphosphate, ER  

derived Ca2+

2. Negative – NADH and acetyl CoA

2. Citric Acid Cycle

a. 9 steps that complete the oxidation of one acetyl equivalent  (two Carbon atoms oxidized to CO2)

i. Each oxidation step accompanied by the release of  

CO2 and the reduction of NAD+ to NADH

ii. Resulting metabolite – succinyl CoA

1. Sufficient free energy to generate one GTP  

and reduce two additional electron carriers –

one FADH2 and one NADH

iii. Final metabolite – oxaloacetate

1. Four Carbon molecule that initiates the cycle

BSC 300-001 Week 6 Notes

iv. How many turns of the Citric Acid cycle does it  take to oxidize all carbons in one pyruvate?

1. Three

b. Steps:

i. 1 - The two carbon acetyl group is condensed with  the four carbon oxaloacetate to form a six carbon  citrate molecule

1. High energy thioester bond of Acetyl CoA  

drives reaction

2. Citrate synthase is a major site of positive  

and negative allosteric regulation

ii. 2 and 3 – Isomerization of citrate

1. Tertiary alcohol of citrate is not readily  

oxidizable, but the secondary alcohol  

isocitrate is

2. Covalent bonds rearranged to make  

metabolite more reactive

a. Creating a molecule with greater  

reduction potential

iii. 4 – generation of CO2 by a NAD+ dependent  dehydrogenase

1. Isocitrate oxidized to the 5 Carbon alpha  

ketoglutarate

2. Step releases one molecule of CO2 and  

reduces NAD+ to NADH

iv. 5 – Another dehydrogenase complex catalyzes  oxidation of alpha ketoglutarate to succinyl CoA 1. Analogous to pyruvate dehydrogenase: large  multiprotein complex uses Coenzyme A to  

generate a high energy thioester bond

2. Reduces NAD+ to NADH

3. A third carbon is completely oxidized – one  full pyruvate equivalent has been oxidized

v. 6 – substrate-level phosphorylation – succinyl-CoA  hydrolysis drives formation of an energy-rich  

phosphate bond

1. Promotes replacement of CoA by an  

inorganic phosphate which is then  

transferred to a diphosphate nucleotide

2. In plants and bacteria, ATP is formed  

directly in this step

3. In animals, GTP is formed and used to  

synthesize ATP by the actions of another  

enzyme

vi. 7 – Flavin-dependent dehydrogenation: succinate  oxidized to fumarate, converting FAD to FADH2

BSC 300-001 Week 6 Notes

1. FAD is a coenzyme similar to NAD+ and  

accepts two hydrogen atoms is reduced to  

FADH2

2. Enzyme succinate dehydrogenase is directly  involved in the electron transport chain, aka  

succinate CoQ reductase

vii. 8 – Hydration of a Carbon-Carbon double bond 1. Fumarate hydrolyzed to form malate

a. Makes metabolite more reactive

b. Rearrangement makes it a better  

reducing agent so it can catalyze  

another step

viii. 9 – TCA cycle is completed with the NAD+  dependent oxidation of malate to oxaloacetate

1. Highly endergonic

2. Moves in the forward direction because the  

exergonic citrate synthase reaction keeps  

oxaloacetate concentrations very low

ix. What you need to know

1. Importance of CoA and the resulting  

thioester bonds in driving reactions forward

a. Unstable high energy bonds that can  

be easily broken to form more stable  

bonds

2. The functional definition of dehydrogenase

enzymes and their importance in reducing  

NAD+ and FAD

3. Citrate synthase as the key regulated enzyme  in this cycle

4. Succinate dehydrogenase as a shared  

enzyme between TCA cycle and ETC

5. The number of NADH, FADH2, and  

ATP/GTP generated per pyruvate or  

pyruvate equivalent

x. Overall 4 Oxidation-Reduction Reactions in the  TCA cycle

1. 2 Carbons released as CO2

2. 3 reactions: NAD+ reduced to NADH

3. 1 ATP equivalent generated

4. 1 reaction: FAD reduced to FADH2

5. At the end of two cycles, we have oxidized  

one glucose

a. We have only made 2 ATP’s (2 in

glycolysis and 2 here)

b. Energy trapped in electron carriers

BSC 300-001 Week 6 Notes

c. Now we figure out how to transfer  

energy out of it

xi. The basic function of the TCA cycle is to produce  

high energy carrier molecules (NADH and FADH2)  

that will be used in subsequent steps during  

oxidative phosphorylation to produce ATP

3. The malate aspartate shuttle

a. NADH generated in cytosol has no utility

i. Cytosolic enzymes that require reducing power use  

the similar reducing agent NADPH

ii. NADH can be converted to NADPH

1. Its electron can be transported into the  

matrix to help power electron transport

system

2. 2 systems of mitochondrial membrane  

shuttle systems drive this process:

a. Glycerol phosphate shuttle –

oxidizes cytoplasmic NADH and  

reduces matrix FAD to FADH2

b. The malate aspartate shuttle oxidizes  

cytoplasmic NADH and reduces  

matrix NAD+

4. Metabolites from other cellular processes fed into the citric acid  cycle and make energy themselves

a. Fats are oxidized in the mitochondria by Beta oxidation to  generate Acetyl CoA

b. Metabolic intermediates of glycolysis and TCA cycle are  substrates for diverse anabolic pathways synthesizing:

i. Amino acids

ii. Nucleotides

iii. Lipids

c. Therefore, TCA cycle is the central metabolic pathway of  the cell

v. Fats

1. Glycerol molecule with fatty acids attached

2. Saturated with hydrogens

3. A lot of reduced energy

4. Stored as triglycerides in lipid droplets in adipose cells

a. Upon glucagon stimulation, fatty acids are cleaved from  glycerol and released to bloodstream

5. Transported into cells and in ATP dependent manner are esterified  to CoA

6. Fatty acyl CoA transported into mitochondrion for energy  extraction via the Beta oxidation pathway

a. Release a lot more energy in the form of NADH and  

FADH2

BSC 300-001 Week 6 Notes

vi. β-oxidation: the Fatty acid cycle

1. 2 dehydrogenase enzymes consecutively oxidize carbons 2 and 3  of the fatty acyl chain

a. Reduces one FAD and one NAD+

2. Another enzyme cleaves acetyl CoA and joins another CoA to the  shortened chain

a. Released acetyl CoA fed into TCA cycle

3. Therefore, each turn of the Fatty Acid Cycle generates:

a. 4 NADH

b. 2 FADH2

c. 1 ATP equivalent

4. Only worry about the general process of β-oxidation

vii. Peroxisomes

1. Membrane-bound vesicles that contain more than 50 oxidative  enzymes that catabolize diverse biomolecules

2. Use O2 to oxidize these substrates, producing hydrogen peroxide  (H2O2) as a bi-product

3. The peroxisomal enzyme catalyase converts this toxic molecule to  O2 and H2O

4. Two critical functions:

a. β-oxidation of very long chain fatty acids (VLCFAs)

i. Fatty acid tails longer than 22 carbons

ii. The shorter FA-CoA molecules are then transported  

to mitochondria

b. Production of Plasmalogens, a lipid enriched in  

cardiovascular and myelin cell – functioning to insulate  

excitable cells

b. The Electron-Transport Chain and Generation of the Proton-Motive Force i. Oxidative Phosphorylation

1. Complex 2 step process:

a. Electron Transport Chain establishes H+ electrochemical  

gradient: proton-motive force

b. Flow of protons drives ATP production via ATP synthase

2. Energy for ATP production provided by electrons in the carriers  NADH and FADH2

a. About 3 ATPs per NADH generated during TCA

b. About 2 ATPs per FADH2

3. Redox Potential – measure of the tendency of a chemical species to  acquire electrons from a specific donor

a. Measured in volts – more positive the Redox potential, the  

stronger the affinity for electrons (greater electron  

acceptor)

i. Electrons pass through ETC from one acceptor to  

another, in each step they move to a molecule with  

more positive Redox potential, ultimately to O2

ii. The Electron Transport Chain

BSC 300-001 Week 6 Notes

1. Electrons associated with either NADH or FADH2 are used to  reduce numerous electron carriers associated with four inner  membrane protein complexes (ETC)

2. Electrons passed through carriers with consecutively greater  positive redox potential

3. Energy releasing reactions coupled to conformational changes in  three of the complexes

a. These changes move protons (H+) across the inner  membrane into the intermembrane space, establishing an  electrochemical gradient – the proton motive force

4. Within these complexes 5 types of electron carriers accept and  donate electrons according to their increasing Redox potentials a. All but ubiquinone are prosthetic groups, non-protein  molecules, strongly bound to the protein complexes

b. Electron carriers:

i. Flavoproteins

1. Polypeptides containing 1 of 2 related  

prosthetic groups

a. FMN (flavin mononucleotide) or

i. Found in Complex I

b. FAD (flavin adenine dinucleotide)

i. Found in Complex II  

ii. This is the electron carrier  

reduced by succinate  

dehydrogenase in the TCA  

cycle

c. Each accepts and donates 2 protons  

and 2 electrons

ii. Cytochromes

1. Possess heme prosthetic groups with iron  

centers

a. Alternate between Fe2+ and Fe3+ by  

gain or loss of a single electron

2. 3 distinct cytochromes in complexes III and  

IV

3. A distinct Cytochrome C peripheral  

membrane protein is water soluble and  

peripherally associated with the inner  

membrane

iii. Three Copper Atoms

1. All associated with Complex IV

2. Accept and donate a single electron as they  

alternate between Cu2+ and Cu+

iv. Iron-sulfur proteins

1. Iron linked to non-heme sulfur centers in I,  

II, and III

BSC 300-001 Week 6 Notes

2. Also accept and donate a single electron

v. Ubiquinone (coenzyme Q: CoQ)

1. Doesn’t have a prosthetic group

a. Lipid soluble due to a long  

hydrophobic tail

b. Capable of rapid lateral diffusion

2. Accepts and donates 2 electrons and protons

a. Partially reduced free radical =  

semiquinone

b. Fully reduced = dihydroquinone  

(CoQH2)

iii. The Process of the Electron Transport Chain

1. Complex I: NADH-CoQ Reductase

a. Principle function – catalyze oxidation of NADH and  

reduction of ubiquinone (CoQ)

b. All prosthetic groups in the soluble domain extend into the  matrix

c. Electrons are transferred from NADH to FMN

i. Then through seven Fe-S centers

d. Final step, the electrons reduce ubiquinone (CoQ) to  

dihydroquinone (CoQH2)

i. 2 protons are absorbed from the matrix

e. Alters conformation of the complex, shifting a horizontal  helix (t helix) that opens 4 channels for active transport of  

protons

f. Alters the ionic state of uniquely positioned residues that  accept and release 4 protons simultaneously into the  

intermembrane space

g. CoQH2 is now primed to deliver electrons to the next  

complex: Complex III: CoQH2-cytochrome c reductase

h. Several other enzymatic processes in the mitochondria  

contribute to available CoQH2

2. Complex II: Succinate CoQH2 reductase

a. The same complex of the TCA cycle that catalyzes  

reduction of FAD to FADH2

b. Feeds lower energy electrons into ETC

c. FADH2 remains physically associated with the complex  and reduces an Fe-S center

d. There is not accompanying proton transfer

e. These electrons also reduce CoQ to CoQH2 which, like  CoQH2 from complex I, delivers them to complex III

3. Complex III: CoQH2-cytochrome c reductase

a. Q cycle enhances the number of protons pumped into  

intermembrane space

i. Maximizes movement of protons from matrix to  

mitochondria

BSC 300-001 Week 6 Notes

b. Oxidizes 2 CoQH2 but reduces one CoQ, so the net effect  

is the movement of 4 protons into the membrane space for  

each CoQH2

c. The Q site near the intermembrane space binds a CoQH2

d. The 2H+ are released into the intermembrane space

e. One electron immediately reduces the a cytochrome c  

peripheral protein

f. The second electron is passed through carriers to a CoQ  

bound to the Q site near the matrix

g. CoQ is released from the Q site

h. A second CoQH2 binds the Q and the process repeats

i. As result of the Q cycle:

i. 4 protons pumped into intermembrane space

ii. Only 1 CoQH2 equivalent is really oxidized

j. Cytochrome c peripheral protein carries 1 electron at a time  

to the final complex

4. Complex IV: cytochrome c oxidase

a. Transports two additional H+ to IM space for every  

electron pair

b. Because it can carry 1 electron at a time, four are required

c. These 4 electrons and protons from the matric are used to  

reduce one oxygen, making two waters

i. This is why Oxygen, the terminal electron acceptor,  

is the driving force of aerobic respiration

c. Harnessing the Proton-Motive Force to Synthesize ATP

BSC 300-001 Week 7 Notes

I. Chapter 13 – Moving Proteins into Membranes and Organelles, Part 1 a. Targeting Proteins to and across the ER Membrane

i. Important Concepts

1. Synthesis of secreted proteins, integral plasma-membrane proteins,  and proteins destined for the ER, Golgi complex, plasma  

membrane, lysosome, and secretion begins on cytosolic ribosomes,  

but is transferred to the ER membrane for completion

2. A signal sequence, SRP, and SRP receptor system docks the  

ribosome on an ER translocon and cotranslationally inserts the  

nascent protein into or through the ER membrane

ii. Overview of major protein-sorting pathways in eukaryotes

1. Typical mammalian cells express about 10,000 different kinds of  

proteins

2. All nuclear-coded mRNAs initiate translation on cytosolic  

ribosomes

a. AKA free ribosomes

b. Signal based – sequences of residues or modifications to  

specific residues directs sorting

c. Can occur during translation of immediately after

d. Vesicle-based trafficking (aka secretory pathway)  

transports proteins from ER to final destination in  

membrane bound vesicles

3. Nonsecretory pathway (right)

a. Proteins with no ER signal sequence are translated on free  

ribosomes

i. Such proteins are cytosol soluble

b. Organelle specific targeting sequences direct delivery to  

and import into the nucleus, mitochondria, chloroplast,  

peroxisomes

i. Other sequences may target to sub-organelle  

locations

4. Secretory pathway (left)

a. Ribosomes initiate protein synthesis in cytosol

b. ER signal sequence (pink) directs ribosome to dock on  

rough ER

c. Nascent proteins (proteins that are still in the process of  

being translated) translocated into ER lumen or is  

embedded in ER membrane

d. ER synthesized proteins transported by vesicles to Golgi  

complex for further processing and packaging for delivery  

to PM, lysosome, ER, or Golgi retention

iii. Endoplasmic reticulum

1. General

a. Continuous network of membranes dispersed throughout  

much of the cytoplasm

b. Flattened sacs called cisternae

BSC 300-001 Week 7 Notes

c. Highly dynamic, undergoing continual turnover and  reorganization

2. Rough ER

a. Continuous with outer membrane of nuclear envelope b. Principle role – synthesis of proteins that will be part of  the endomembrane system or secreted

c. Covered with ribosomes (complexes that translate mRNAs  into proteins) on its cytosolic surface for protein translation 3. Smooth ER

a. Continuous with Rough ER

b. Functions:

i. Synthesis of steroid hormones in endocrine cells,  

such as in gonad and adrenal cortex

ii. Detoxification of various organic compounds in the  liver through action of Cyt P450 enzyme family

iii. Sequestration of Ca2+ from cytoplasm

1. Regulated release controls function of many  

cytosolic proteins

2. Ex: in muscle cells the SER (sarcoplasmic  

reticulum) controls the release of Ca2+ to  

regulate muscle contraction

4. Both the rough and smooth ER are responsible for new lipid  synthesis, but in cells with heavy protein production, and therefore  extensive ribosomes on the RER, this job is primarily performed  by the SER

5. Study of Cytomembranes – The Pulse Chase Experiment a. Autoradiography – uses radioactively labeled materials  and exposure to photographic film to analyze diverse  

biological processes

b. Combined with subcellular fractionation (lysing cells and  separating organelles so that they can determine parts of the  cell) this was an invaluable technique in determining the  order of protein synthesis and trafficking through the  

endomembrane system

c. Fed cells media with radioactive amino acids

i. Remove media

ii. Add normal media

iii. Cells producing small initial amino acids and then  chased with normal amino acids

iv. Cytopreservation – flash freezing

d. Showed that at different times after the pulse, the  

radioactively labeled proteins were found in different parts  of the cell

e. It does not tell us anything about how cells know the  correct destination

6. Synthesis of Proteins on Membrane-Bound versus Free Ribosomes

BSC 300-001 Week 7 Notes

a. About 1/3 of polypeptides encoded by human genome  synthesized on RER ribosomes:

i. Secreted proteins, integral membrane proteins, and  soluble proteins of organelles

b. Polypeptides synthesized on “free” ribosomes include: i. Cytosolic proteins, peripheral membrane proteins,  nuclear proteins, and proteins destined for  

chloroplasts, mitochondria, and peroxisomes

c. Free or RER synthesis determined by N-terminal amino  acid sequences

i. All translation begins in the cytoplasm on free  

ribosomes

ii. N-terminus is the first end to be translated

iii. Proteins destined for RER synthesis possess an N terminal signal sequence that directs the transfer of  

the ribosome/mRNA/peptide complex to the RER

iv. Polypeptide is translated into ER cisternae space  through protein-lined pore

v. A few proteins have the signal sequence at the C

terminus and are thus mostly translated before they  

are delivered to the RER

7. Cotranslational translocation – Synthesis of secreted proteins a. Messenger RNA bound by free ribosome in cytoplasm b. Hydrophobic N-terminal signal sequence (16-30 residues)  

on nascent protein is recognized by protein called signal  recognition particle (SRP)

c. SRP binds signal sequence and docks to SRP receptor i. Association between SRP and receptor is

accomplished via cooperative binding to GTP

d. Associated with SRP receptor is translocon complex (Sec  61 complex) that mediates RER protein translocation into  the lumen

e. GTP hydrolysis disengages SRP from receptor

i. Nascent peptide is fed into the open translocon  

channel and remains associated with hydrophobic  

residues

ii. Translocation driven entirely by translation  

elongation by ribosome

f. Once peptide is partially translated into RER lumen, signal  sequence is cleaved by signal peptidase

g. Chaperones assist in proper folding

h. Because extracellular space is oxidizing environment,  secreted proteins (and extracellular regions of integral  membrane proteins) are strengthened by si-sulfide bridges  – bonds catalyzed by the protein disulfide isomerase (More info later in lecture)

BSC 300-001 Week 7 Notes

8. ATP hydrolysis powers post-translational translocation in some  secretory proteins of yeast

a. Post-translational translocation is common in yeast and  occasionally in other higher eukaryotes

b. SRP and bound ribosome not involved, but translocon  (Sec61) is the same

c. Freely translated, unfolded protein contains N-terminal  signal sequence

d. Random association between signal sequence and  translocon allows association, but after signal sequence is  cleaved peptide can randomly slide in or out

e. Unidirectional movement of nascent peptide into lumen  driven by the chaperone Bip

i. Bip binds peptide in ATP dependent manner and  

ATP hydrolysis is catalyzed by Sec63 protein  

(associated with the translocon)

ii. Repetitive Bip binding pulls peptide into lumen

iii. Uncertain whether similar process occurs for some  proteins in higher eukaryotes

9. Classes of ER membrane proteins

a. Rough ER synthesizes five topological classes of integral  membrane proteins as well as GPI anchored proteins

b. Topology – number and orientation of membrane spanning  segments

c. Integral membrane proteins

i. Classified by orientation in the membrane, locations  of N- and C- termini, and the types of targeting  

signals used to direct/orient them

ii. Hydrophobic alpha helices – embedded in the  

membrane bilayer

iii. Hydrophilic regions – fold into various  

conformations outside the membrane

d. Type I-III and tail-anchored are all single pass TM proteins i. Each has a specific orientation in membrane with  

N- or C-terminus facing cytosol

1. Type I – C-terminus facing cytosol

2. Type II – N-terminus facing cytosol

3. Type III – C-terminus facing cytosol

4. Tail-anchored protein – C-terminus facing  

cytosol

ii. Different mechanisms for recognizing the nascent  proteins and orienting the transmembrane domains

e. Type IV proteins – multiple transmembrane alpha helices  (aka multipass membrane proteins)

i. G protein-coupled receptors

BSC 300-001 Week 7 Notes

1. Seven alpha helices, N-terminus is  

exoplasmic, and C-terminus is cytosolic

f. GPI-anchored proteins are always anchored by their C

terminus and face the exoplasm

b. Insertion of Membrane Proteins into the ER

1. Membrane Insertion and Orientation of Type I single-pass transmembrane  proteins

a. Simplest mechanism – N-terminus is exoplasmic and single TM pass i. Translocation initiation and signal sequence cleavage by same  mechanism as soluble secreted proteins

ii. Nascent peptide elongates and is pushed into ER lumen

iii. Elongation continues until hydrophobic transmembrane stop transfer anchor sequence (STA) enters translocon – prevents  

nascent chain from extruding farther into ER lumen which  

adopts alpha helical secondary structure

iv. Translation continues with C-terminus remaining in cytosol

v. Translocon is hinged, like a clam, and oscillates between open  and closed states, exposing a hydrophobic cleft

vi. Stop-transfer anchor sequence moves laterally through this  cleft and diffuses into hydrophobic interior of phospholipid  

bilayer

vii. Translation stops at stop codon

1. Ribosomal subunits released into cytosol

2. Nascent protein is free to diffuse laterally in the ER  

membrane

2. Membrane Insertion and Orientation of Type II and Type III single-pass  transmembrane proteins

a. Type II and III proteins lack N-terminal signal sequence

i. Possess single internal hydrophobic signal-anchor sequence

(SA) – serves as ER signal sequence and transmembrane alpha

helix

b. Type II proteins – N-terminus is cytoplasmic

i. SRP binds internal signal-anchor sequence and associates with  SRP receptor on ER membrane

ii. Recall PI and PS have negatively charged head groups and are  enriched in cytosolic leaflet

iii. Signal-anchor sequences are flanked by a short stretch of  

positively charged residues which orient the alpha-helix within  

the membrane

iv. For type II proteins, the positive residues are N-terminal to the  signal-anchor sequence

v. Elongation extrudes remainder of nascent protein into ER  

lumen

vi. Translation extrudes C-terminus into lumen

vii. There is no cleavage of signal sequence

BSC 300-001 Week 7 Notes

c. Type III proteins – N-terminus is exoplasmic, but no cleaved signal  sequence

i. Short N-terminus prior to signal anchor sequence

ii. Positively charged residues are to C-terminal to signal-anchor  sequence effecting the opposite orientation

iii. C-terminal elongation completed in cytosol

iv. Ribosomal subunits are released

v. Orientation depends on which side of the step anchor sequence  the terminus is on

1. Positively charged residues adjacent to signal-anchor  

sequence

a. Experimentally moving charged residues to  

opposite side of TM domain of type II or III  

proteins will invert their orientation in  

membrane

3. Insertion of C-terminal tail-anchored proteins

a. No N-terminal signal sequence

b. Hydrophobic C-terminal anchor sequence

i. Not available for membrane insertion until translation is  

complete and protein is released from free ribosome

ii. Transfer protein Get3, rather than SRP, binds hydrophobic C terminal and in ATP dependent manner docks on distinct ER  

membrane receptor complex: Get1/Get2

1. ATP hydrolysis promotes transfer of C-terminus to  

Get1/Get2, which facilitates C-terminal diffusion into  

membrane

4. Topogenic sequences determine orientation of Type IV multipass  transmembrane proteins

a. Type IV proteins can orient with N-terminus either exoplasmic or  cytoplasmic

i. No signal sequence

ii. Orientation of N-terminus determined by positively charged  residues flanking first internal signal anchor sequence

iii. Alternating SA and STA sequences maintaining alternating  orientation of TM domains

1. Type A fed directly into lumen and then there is a stop  

anchor

5. GPI-anchored proteins

a. Synthesized the same as Type I TM proteins

i. N-terminal signal sequence (cleaved) and internal stop transfer  anchor sequence usually near C-terminus

ii. Amino acid sequence in lumen adjacent to TM domain  

recognized by GPI transamidase proteins

1. Catalyzes cleavage and covalent transfer of luminal  

portion of protein to GPI anchor

b. Benefits of GPI anchor:

BSC 300-001 Week 7 Notes

i. Increased diffusion rate in leaflet

ii. Cleavable for release of protein to exoplasm

iii. Targeting to lipid rafts and/or apical surface of polarized cells 6. Synthesis of the core portion of an oligosaccharide

a. N-linked glycosylation in the RER

i. Nearly all ER synthesized proteins are extensively modified via  glycosylation

1. Such modifications catalyzed by destination-specific  

glycosyltransferase enzymes

ii. Ultimate composition and arrangement of sugars  

(glycosylation) for any protein is both cell-type and organelle  

specific

1. In different cell types, the same protein can have  

distinct glycosylation patterns

iii. These modifications alter protein conformation, solubility,  participate in inter- and intracellular signaling, protein-protein  interaction and cell-cell recognition

b. Biosynthesis of the oligosaccharide precursor

i. All proteins originating in ER are glycosylated with a core  chain of 14 sugar monomers  

1. Assembled and covalently linked to the lipid dolichol  

phosphate

ii. Composition and orientation of these sugars identical or all ER  processed proteins: 2 NAD, 9 mannose and 3 glucose  

monomers assembled in a trident pattern

iii. Assembly begins on ER cytoplasmic leaflet the glycoplipid is  flipped to the ER lumen, where glycosylation is completed

iv. Glycocosyltransferase enzymes catalyze transfers via  

hydrolysis of phosphorylated nucleotides covalently attached to  the sugar monomers

v. Called N-linked glycosylation because completed sugar chain  is covalently transferred to specific asparagine (symbol N)  

residues in the sequence Asp-X-Ser/Thr (X is any amino acid)

vi. Not all Asp-X-Ser/Thr sequences are glycosylated and its  unknown how they are targeted

vii. Transfer of chain to this sequence is catalyzed by  

oligosaccharyltransferase

viii. Antibiotic tunicamycin blocks assembly of dolichol phosphate  and inhibits this critical glycosylation from occurring,  

impeding bacterial growth

ix. Terminal 3 glucose residues serve as a signal that the sugar  chain is mature and ready to be transferred to a protein

1. These sugars play an important role in quality control  

and ensuring ER proteins adopt correct 3D  

conformation

BSC 300-001 Week 7 Notes

2. If properly folded, the terminal 3 glucose sugars are  

removed and the protein is ready for export to the Golgi

c. Protein Modifications, Folding, and Quality Control in the ER i. Action of protein disulfide isomerase (PDI)

1. Secreted proteins and extracellular domains of TM proteins face a  harsh extracellular oxidizing environment, which can alter protein  conformation and function

a. Such proteins are stabilized by intramolecular disulfide  

bonds between sulfhydryl groups of cysteine residues

i. Formation of disulfide bridges (which is what  

protein disulfide isomerase does)

2. Protein Disulfide Isomerase – serves as the ER luminal oxidizing  agent that catalyzes generation of disulfide bonds in substrate  

proteins

3. Regeneration – reduced PDI is subsequently oxidized by the ER  protein Ero1 (ER Oxioreductin) to keep a supply of oxidized  

isomerase in ER lumen

ii. Chaperones and other ER proteins facilitate folding and assembly of  nascent proteins

1. BiP chaperone proteins bind nascent peptides as they translocate  into ER, preventing improper folding and aggregation

2. PDI also participates in proper folding through formation of di sulfide bridges

3. Calnexin and calreticulin bind modified oligosaccharide chains of  peptides that have been identified as misfolded, preventing  

inappropriate intramolecular association of hydrophobic residues  (ER Quality Control)

4. Peptidyl prolyl isomerases promote cit to trans isomerization of  specific proline residues to accelerate rotation around peptidyl

prolyl bonds in unfolded peptide segments – often the rate limiting  step in protein domain formation

iii. ERAD and Quality Control

1. After transfers to the polypeptide, the core oligosaccharide will be  extensively modified

2. First part of this regulates a process that ensures proper folding of  all ER synthesized proteins: the ER quality control system

a. Enzymes called glucosidases cleaves 2/3 of terminal  

glucose monomers and the monoglucosylated polypeptide  

is bound by chaperones calnexin and calreticulin

i. These chaperones promote proper folding by  

preventing aggregation and premature export from  

the ER

3. After successive rounds of attempting to properly fold a protein it  is considered defective and target for destruction

a. ER enzymes named alpha-mannosidases trim 3-4  

mannoses, resulting in carbohydrate moiety that is

BSC 300-001 Week 7 Notes

recognized b the protein OS-9 and targeted for dislocation  

from ER lumen and degradation in cytosol

b. Protein complex called ER Associated Degradation  

complex (ERAD) facilitates proposed pulling of misfolded  

proteins through ER membrane

i. Very slow and very efficient

ii. In the cytosol ERAD associated ubiquitin ligases  

tag the misfolded protein for degradation by  

proteasomes

iv. The Unfolded Protein Response

1. Under conditions of extreme stress, misfolded proteins can be  

generated faster than the ERAD system can process them

a. Triggers Unfolded Protein response

i. Halts protein translation to prevent accumulation of  

misfolded proteins

ii. Degrades misfolded proteins

iii. Activates signaling pathways that lead to increased  

production of ER chaperones

iv. If a cell cannot do this, it will kill itself

2. Membrane associated protein sensor Ire1 is normally kept inactive,  through association with BiP chaperones

a. Accumulation of many unfolded proteins sequesters BiP  

chaperones, releasing Ire1 and allowing it to dimerize and  

become active

i. Active Ire1 is an endonuclease, which functions to  

process an unspliced mRNA from the gene Hac1

1. Once spliced Hac1 mRNA is translated,  

generating a transcription factor that  

activates transcription of genes that encode  

chaperones and other proteins involved in  

protein folding

3. In mammals a second ER membrane sensor protein ATF6 is also  

activated by regulated cleavage

a. Active ATF6 is also a transcription factor that translocates  

to the nucleus to promote transcription of ER chaperones

i. If such Ire1 and ATF6 regulated protein cannot re

establish normal ER activities they activate pro

apoptotic proteins that initiate cell death in order to  

remove the damaged cell

II. Moving Proteins into Membranes and Organelles, Part 2

a. Targeting of Proteins to Mitochondria and Chloroplasts

i. Protein import to mitochondria and chloroplasts

1. Mitochondrial and chloroplast encoded proteins are translated by  

organelle encoded ribosomes

a. A large amount of these proteins are nuclear encoded and  

must be transported from the cytosol

BSC 300-001 Week 7 Notes

b. Translated on free ribosomes but remain unfolded

i. Chaperones

ii. Unfolded proteins transported via sequential  

membrane protein complexes

2. Evolutionarily shared import mechanisms

a. Energy dependent process

b. Occurs at points where outer and inner membranes make  contact

c. Two membrane bound transport systems:

i. Outer membrane

1. Directs target protein to intermembrane  

space

ii. Inner membrane

1. Directs organelle to correct suborganelle  

compartment (membrane or matrix)

ii. Protein import to nucleus

1. The nuclear envelop possesses large transport complexes called  nuclear pores that span the inner and outer nuclear envelope  membranes

a. Selective gates actively transport specific macromolecules  but also allow passive diffusion of smaller molecules

b. Pores are large enough to allow native conformation  

proteins to be imported

iii. Signal sequences direct proteins to correct compartment 1. Primary sequence of many nuclear and ER translated proteins  contains specific signal sequences

a. Sorting signals comprised of unique amino acid sequences  are recognized by other cellular proteins and used as a  

mailing address for delivery or retention

b. Typically stretch of 4-60 amino acids

c. Often but not always cleaved from the mature protein it has  reached its destination

iv. Proteins unfold to enter mitochondria

1. Amphipathic N-terminal targeting sequences direct proteins to  mitochondrial matrix

a. Matrix targeting sequence

2. 20-50 aa in length: rich in hydrophobic, basic and polar, but  typically lack acidic residues

3. Adopt alpha-helix conformation with positively charged residues  on one side of the helix and hydrophobic residues on the other 4. Amphipathicity is critical as mutations that alter this character  inhibit mt targeting

v. Generalized protein import into mitochondrial matrix

1. No analog of ER Signal Recognition Particle

2. Matrix targeting sequence can interact directly with mt outer  membrane import receptor

BSC 300-001 Week 7 Notes

3. Genetic screens have identified different receptors for different  classes of mt proteins

4. Nascent proteins kept unfolded via ATP dependent activity of  cytosolic chaperones

5. Import receptors then transfer nascent protein to a general import  pore, composed mainly of the TOM40 protein (translocon of the  outer membrane)

6. Contact sites between outer and inner membranes (MAMs)  establish link between TOM40 and diverse translocon on inner  membrane channels (TIMs)

a. TIMs are specific to types of matrix targeting sequences  and coupe translocation with targeting the nascent protein i. Ex: proteins bound for matrix have MTS recognized  and bound by the TIM23/17 complex

7. As peptide emerges into matrix, protease cleaves the MTS 8. Matrix HSP70 anchored to TIM complex by another TIM protein  pull nascent peptide intro matrix via ATP hydrolysis (like post translational translocation in yeast ER for some proteins) 9. In the matrix other chaperones facilitate proper folding 10. Three energy inputs required for import:

a. Cytosolic Hsp70 – expends ATP energy to maintain  recursor proteins in an unfolded state for translocation  through translocons

b. Matrix Hsp70 anchored to the Tim44 protein – may acts as  a molecular motor to pull the protein into the matrix

c. H+ electrochemical gradient (proton-motive force) across  the inner membrane – matrix negative charge may draw  proteins due to positive residues in MTS

11. Imported mitochondrial membrane proteins

a. Proteins destined for inner membrane, intermembrane  space, or outer membrane – have one or more additional  targeting sequences

i. And one of a number of mechanisms are used for  

translocation

12. Three pathways to inner mitochondrial membrane from the cytosol a. ATS delivers unfolded protein to different IM TOM  complex

i. Resident IM proteins possess STA sequence that is  recognized as it translocates

ii. As with ER proteins, STA directs integration into  inner membrane

b. Some inner membrane proteins possess a unique sequence  recognized by the mt protein Oxa1

i. Folded peptides translocate entirely into matrix and  Oxa1 integrates their TM domains into IM

BSC 300-001 Week 7 Notes

c. Multipass TM proteins lack N-term MTS but have multiple  

internal mt targeting sequences

i. TOM/TIM complexes distinct for multipass protein  

promotes transfer and insertion

ii. Similar to ER SA and STA sequences

13. Two pathways to mitochondrial intermembrane space

a. Path A predominates

i. Ex: Cytochrome b2

ii. N-term matrix-targeting sequence – cleaved by  

matrix protease

iii. Stop transfer sequence

1. Blocks translocation of the protein across  

the inner membrane vis Tim23/17

iv. Inner membrane protease cleaves protein on the  

intermembrane side of the stop-transfer sequence

b. Path B: specialized pathway for cysteine rich proteins  

stabilized by disulfide bonds

i. No N-terminal matrix-targeting sequence or Tim  

targeting sequence

ii. Rather, TOM40/Mia40 recognize cysteine residues  

in nascent protein (with Cx3C or CX9C motifs)

iii. Mia40 and Erv1 coordinate formation of disulfide  

bonds in the proteins

iv. Which maintains conformation and prevents reverse  

translocation through Tom40

b. Targeting of Peroxisomal Proteins

i. Peroxisomes

1. Single membrane organelles containing diverse oxidative enzymes  (oxidases) that use molecular oxygen (O2) to remove hydrogens  

from organic substrates (ex: VLFAs)

2. Products are oxidized substrates and peroxide (H2O2)

a. !"# + %# → ! + "#%# 

3. H2O2 is toxic but is used by another peroxisome enzyme, catalase,  to oxidize other substrates like phenols, formaldehyde, alcohol,  

and formic acid

4. This yields the oxidized substrate and H2O

a. "#%# + !"# → ! + 2"#%

ii. PTS1-directed import of peroxisomal matric proteins

1. Peroxisome luminal proteins:

a. All encoded by nuclear genes

b. Synthesized on free ribosomes in the cytosol

c. C-terminal peroxisomal-targeting sequence PST1  

(SKL/Ser-Lys-Leu sequence)

d. PTS1 recognized by cytosolic receptor – targets proteins  

for transport to peroxisome lumen

2. Peroxisomal import system can translocate folded protein

BSC 300-001 Week 7 Notes

3. PTs bound by soluble receptor Pex5

4. Pex5 joins the peroxisome membrane complex Pex14 allowing  release of cargo protein into matrix

5. Disassembly of Pex5/Pex14 complex driven by  

monoubiquitination of Pex5 – alters association but does not  

promote Pex5 degradation

iii. Model of peroxisomal biogenesis and division

1. De novo formation of peroxisomes:

a. First stage – incorporation of peroxisomal membrane  

proteins into precursor vesicles derived from ER

i. Pex 19 – receptor for membrane targeting  

sequences

ii. Pex 3 and Pex 16 complex – required for proper  

insertion of proteins into the forming peroxisomal

membrane

b. Peroxisomal membrane protein insertion – produces a  

peroxisomal ghost, which imports matrix-targeted proteins

i. PTS1 and PTS2-bearing matrix proteins – imported  

via Pex cytosolic receptors

2. Proliferation – most peroxisomes arise from Pex11-dependent  division of mature peroxisomes

c. Transport into and Out of the Nucleus

i. Pore perforate nuclear envelopes in all eukaryotic cells – passage between  cytosol and nucleoplasm

ii. Each pore is an enormous assemblage of many copies of 30 different  proteins called nucleoporins that collectively create the nuclear pore  complexes (NPC)

iii. Structural and membrane nucleoporins form a wide ring that joins the  outer and inner membranes  

iv. Filaments extend into cytoplasm while similar filaments projecting into  nucleoplasm are bundled into a “nuclear basket”

1. These filaments are thought to aide in import specificity, but some  experiments have shown them to be dispensible

v. FG nucleoporins contain repeats of hydrophobic amino acids [Phe (F) and  Gly (G)] interspersed among hydrophilic amino acids

1. The FG nucleoporins project into the pore and associate via the FG  repeats – establishing a net through which only small molecule and  ions can diffuse

vi. Molecules larger than 40 kDa require assistance of nuclear transport receptors that interact with transported molecule and destabilize FG repeats

vii. Import or export requires targeting sequences: nuclear-localization signal  (NLS) and nuclear-export signal (NES)

viii. Several unique NES and NLS translocates through nuclear pores bound to  nuclear transport receptor

BSC 300-001 Week 7 Notes

1. Transient interactions between receptors and FG-repeats allow  rapid diffusion of nuclear transport receptor-cargo complexes  through the central channel of the NPC

ix. Cargo can only move through nuclear pores when bound to escort protein  called importin

x. Unidirectional movement driven by generation of gradients of  Cargo/importin complexes that is high on the cytoplasmic side of the  nuclear envelope

xi. Importin is recycled back to the cytoplasm through association with asmall  G protein called Ran

xii. Ran hydrolysis of GTP in the nucleus and exchange of GDP for GTP n the  cytoplasm maintains these gradients that faceilitate movement into the  nucleus

xiii. Recall G proteins are activation switches for other proteins, they other  proteins, they bind and activate target proteins only when association with  a small G protein called Ran

xiv. Ran hydrolysis of GTP in the nucleus and exchange of GDP for GTP in  the cytoplasm maintains these gradients that facilitate movement into  nucleus

xv. Recall G proteins are activation switches for other proteins, they bind and  activate target proteins only when associated with GTP

1. They turn themselves off through hydrolysis of bound GTP to  GDP

2. GTPase Activating Exchange Proteins (GAPs) – catalyze GTP  hydrolysis, therefore inhibit G proteins  

3. Guanine nucleotide Exchange Factors (GEFs) – catalyze exchange  of GDP for GTP, therefore activate G proteins

4. Active G proteins adopt a conformation that allows them to  interact with their targets and effect some activity or function xvi. Nuclear import: Concentration gradients and permeability 1. Proteins with an exposed NLS are bound by the nuclear transport  receptor importin

2. Importin/cargo complex binds filaments on nuclear pore and is  drawn into FG-nucleoporin network

a. How rapid diffusion into nucleus occurs is not well  

understood

3. In the nucleus, importin/cargo complex binds Ran-GTP which  induces conformational change in importin allowing release of  cargo

4. This produces the concentration gradient of importin/cargo that  drives unidirectional diffusion into nucleus

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