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Exam 2 Study Guide Advanced Cell Bio

by: Izabella Nill Gomez

Exam 2 Study Guide Advanced Cell Bio BCMB 311

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Hey guys! Sorry to post as such a last minute, but I was having trouble finding a set of notes! here is a compilation of everything needed for our second exam in bio 311! I hope you like it!
Advanced Cellular Biology
Dr. Barry Bruce, Dr. J. Park
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This 26 page Study Guide was uploaded by Izabella Nill Gomez on Thursday March 3, 2016. The Study Guide belongs to BCMB 311 at University of Tennessee - Knoxville taught by Dr. Barry Bruce, Dr. J. Park in Spring 2016. Since its upload, it has received 34 views.


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Date Created: 03/03/16
Advanced Cell Bio Chapter 12 Notes! The vast majority of the molecules cannot simply diffuse through the plasma membrane like CO2, O2. Transfer depends on membrane transport proteins that span the lipid bilayer, providing private passageways that can access the membrane for selected substances. Transporters shift small organic molecules or inorganic ions from one side to the other by changing shape. Channels form tiny hydrophilic pores across the membrane through which such substances can pass by diffusion. Most only allow inorganic ions to pass through (ion channels). Can create voltage across the membrane. Hydrophilic molecules pass by the bilayer slowly through simple diffusion, can be accelerated by facilitated transport. The smaller the molecule, the more nonpolar, the faster it will diffuse. Many molecules of interest to cells are polar and water soluble. Solutes-- substances that are dissolved in water--unable to cross the bilayer without the aid of membrane transport proteins: 1. Small nonpolar--diffuses readily if small (H2), ethanol, glycerol not so fast). Glucose rarely crosses. 2. Lipid bilayers are highly impermeable to all charged molecules-- inorganic ions--no matter how small. Because cell membranes are impermeable to inorganic ions, living cells are able to maintain internal ion concentrations different from concentrations of ions in media that surrounds them. Most important inorganic ions: Na+, K+, Ca2+, Cl-, H+. Movement of these plays an essential part in biological processes; most striking is ATP production and nerve cell communication. Na+ most plentiful positive charge ion outside the cell, K+ abundant inside. To avoid being torn apart, the number of positive charge inside (outside) must equal the negative charge inside (outside), the same for fluid. Na+=Cl-, K+=other ions (nucleic acids, proteins, etc.). Tiny excesses of charge around the plasma membrane occurs, imbalances create voltage differences across the membrane called membrane potential. When the cell is “unstimulated”, exchange of cations/anions across the membrane are balanced. Resting membrane potential holds steady, but is not zero (for animal cells, can be between -20-- -200 mV depending on the organism). Negatively charged inside more than outside. Allows transport of metabolites and provides cells excitable with means to communicate with neighbors. Membrane transport proteins occur in many forms and are present in all cell membranes. Each provides a private portal across the membrane for a small solute molecule-- ion, sugar, amino, etc. Each membrane has its own characteristic set of transport proteins. 2 classes of membrane transport proteins and transporters and channels--differ in the way they discriminate between solutes, transporting some but not others. Channels discriminate mainly on the basis of size and electric charge: when open, any ion/molecule small enough and has the right charge passes through. Transporter transfers only molecules/ions that fit into specific binding sites on the protein. Transporters and solutes with great specificity, in same way enzyme binds its substrate, and gives requirement selectivity. What controls what molecules pass into the cell/organelle is concentrations of solute on either side of the membrane. The molecule will spontaneously flow downhill from a region of high concentration to a region of low concentration provided a pathway exists --passive because they need no additional driving force. If a solute at a higher concentration outside the cell, and appropriate channel/transport present, solute will move into the cell via passive transport, without using energy of transport protein. To move against the gradient, a transport protein must do work--drives flow uphill by coupling to some other process that provides an input of energy-- active transport--carried out by pumps. For uncharged molecules passing through determined only by concentration gradient. But few electrically charged molecules, additive force--membrane potential exerts force on any molecule that carries electric charge. Cytosolic side of the plasma membrane is usually at a negative potential relative to the extracellular side, so membrane potential bends to pull positively charged solutes into the cell and drive negatively charged ones out. At the same time, a charged solute will also tend to move down concentration gradient--net driving force of the concentration gradient and membrane potential--electrochemical gradient-- determines the direction that each solute will flow across a membrane by passive transport. For some ions, voltage and concentration gradient work in the same direction (steep electrochemical gradient)--Na+. Not so much for K+. Some cells contain aquaporins in the plasma membrane to facilitate the flow of H2O (slow). Total concentration of solute particles inside the cell-- osmolarity--generally exceeds solute concentrations outside. The resulting osmotic gradient tends to pull H2O into the cell--flow from low solute concentration (high H2O concentration) to high solute concentration (low H2O concentration) called osmosis--if it occurs without constraint, it can make the cell swell. Most animal cells have a gel-like cytosol that resists osmotic swelling. Some amoebae eliminate excess H2O using vacuoles that discharge extra H2O outside. Plants prevent swelling with thought cell walls that can stand strong osmotic differences--makes use of turgor pressure to keep cell walls tense and the stem of a plant rigid--if lost, the plant begins to wilt. Transporters are responsible for the movement of most small, water-soluble, organic molecules and some inorganic ions across cell membranes. Each is highly selective, often transporting only one type of molecule. To guide/propel complex traffic into/out, between different membrane- enclosed organelles, each cell membrane contains a characteristic set of different transporters appropriate to that particular membrane. Ex: plasma membrane has transporters that import amino acids, sugars, nucleotides; lysosome contains H+ transporter to acidify the lysosome and others that have move digestion into the cytosol; the mitochondria has pyruvate imported to use as fuel for ATP generation. Glucose transporter mediates passive transport. The protein crosses the plasma membrane 12 times, can have different conformations, switches randomly between them. In one, a transporter exposes binding sites for glucose to the exterior of the cell; in another, to the interior. Because glucose is uncharged, the chemical component of electrochemical gradient (E.G.) is zero--determined by the concentration gradient alone. Transporters are selective; use only D-glucose because it cannot use L for glycolysis--cells can’t rely on passive transport alone--active transport is essential to maintain the right ionic composition of cells and to import cells at a lower concentration outside the cell than inside--depend on transmembrane pumps, which can carry out active transport in 3 main ways--ATP-driven hydrolyzes ATP to drive uphill transport, coupled pumps link uphill transport of one solute to the downhill of another, light-driven pumps are found mainly in bacteria and use energy from sunlight to drive uphill transport. Different forms of active transport are often linked. ATP driven Na+ pump drives Na+ out of the cell against E.G., then, it is brought back in down E.G. Influx of Na+ provides energy for the transport of other substances into the cell against the E.G. gradient. ATP-driven Na+ pump bas a central role in active transport of small molecules across the plasma membrane of animal cells. Plants, fungi, bacteria use ATP-driven H+ pumps analogously. ATP- driven Na+ pump plays a central part in energy economy og animal cells that typically accounts for 50% or more of ATP consumption. Pump uses energy derived from ATP hydrolysis to transport Na+ out of the cell as it carries K+ in. Pump is known as Na+-K+ ATPase of Na+-K+ pump. Energy from ATP hydrolysis involves a series of proton conformational changes that drive Na+/K+ ion exchange. As part of the process, P group from ATP gets transferred to pump itself. Ion transport involves reaction cycle with each step dependent on the last. Toxin ciabain prevents the pump from binding extracellular K+. Process takes 10 milliseconds . Avoids useless ATP hydrolysis. Na+ functions like a bilge pump in a leaky ship, expelling Na+ constantly entering the cell. This way, the pump keeps Na+ in the cytoplasm at about 10-30 times lower than in the extracellular fluid and K+ 10-30 times higher. Steep concentration gradient of Na+ across the plasma membrane acts with the membrane potential to create a large Na+ E.G., which pulls Na+ back into the cell. High concentrations of Na+ outside the cell on the uphill side of the E.G. is like a large volume of water building behind a dam; large store of energy. Ca2+ is kept at a low concentration in the cytosol occupied with concentration in the extracellular fluid, but less plentiful than Na+ both inside/outside of cells. Ca2+ can bind tightly to a variety of protons in the cell, altering activities. Influx of Ca2+ into the cytosol through Ca2+ channels is used by different cells as a trigger to cell processes (ex: inside concentration, nerve cell communication). If there is a lower concentration of free Ca2+, more sensitive the cell is to increase in the cytosol of Ca2+. Huge concentration difference is achieved by ATP-driven Ca2+ pumps in the plasma membrane and ER membrane. Ca2+ pumps work like Na+, main difference is that Ca2+ returns to its original conformation without the requirement for binding and transporting an ion. Ca2+ and Na+ are similar with a common evolutionary origin. Coupled pumps couple movement of one inorganic ion to that of another movement of inorganic ion, small molecule, etc. If the pump moves both solute in the same direction across the membrane, it is called symport. If it moves in opposite directions called antiport. Transporter that ferries only one type of solute is called a uniport (ex: glucose transporter). Symports that make use of the inward flow of Na+ down a steep E.G. have important role of driving import of other solutes into animal cells. Ex: epithelial cells that line the gut pump glucose from gut lumen to gut epithelium and ultimately to blood). If the cells had only passive glucose import, would release glucose into the gut after fasting as freely as they take in after a feat. Gut epithelials also have glucose-Na+ symport, which allows to take up glucose from the lumen even when the concentration is higher in the cytosol than the lumen. Because Na+ is pumped in, glucose dragged into the cell with it. Both molecules must be present for couple transport to occur. Cells need other transporters to take the glucose out--at apical domain, have Na+-glucose symport--at lateral domain, have passive glucose uniports--which release glucose down the concentration gradient. Na+-H+ exchanger (antiport) in the plasma membrane of animal cells uses downhill influx of Na+ to pump H+ out of the cell--one of the main devices used to control pH in the cytosol. Plants, bacteria, fungi (yeasts) don’t have Na+ pumps in the plasma membrane--rely on H+. Gradient created by H+ pumps in the plasma membrane that pump H+ out, setting up an electrochemical proton gradient across the gradient and creating an acidic pH. In some photosynthetic bacteria, H+ is created by light-driven H+ pumps (ex: beta-rhodopsin). In others, use ATP hydrolysis to pump H+ out of the cell. Turbine-like pumps in lysosomes/vacuoles actively transport H+ from cytosol to organelles, helping keep the pH of the cytosol neutral and the pH of the organelle acidic--crucial to function. Channels allow small H2O soluble molecules to cross from one side of the membrane to the other through hydrophilic channels--through transmembrane pores to allow passive movement of small H2O solubles in/out of the cell. A few channels form large, aqueous pores--ex: proteins that form gap junctions between 2 adjacent cells--porins that form pores in the outer membrane of the mitochondria and some bacteria. If directly connected to the cytosol, would be disastrously leaky--most are narrow, highly selective pores. Ex: aquaporins allow passive diffusion of uncharged H2O--prohibiting movement of ions. Ion channels show ion selectivity, permitting some inorganic molecules through but not others--depends on the diameter and shape of ion channel and the distribution of charged amino acids that line it. Each ion is surrounded by a small shell of H2O that shed to allow ions to pass. Ion channels are not continuously open. Most are gated--specific stimulus triggers them to switch between closed and open states by changing conformation. Does not need to undergo conformational changes with each ion it passes, and so has a large advantage over a transporter with respect to the maximum rate of transport 1000x greater. But cannot couple ion to energy source for active transport. Changes of membrane potential are the basis for electrical signaling in many cells--mediated by alterations in permeability of membranes to ions. In an animal cell, unstimulated or _resting+, negative charges on organic molecules are largely balanced by K+. K+ is actively imported. Plasma membrane also has K+ leak channels that randomly flicker between open/closed states no matter the conditions--when open, K+ has a tendency to flow out of the cell down a steep concentration gradient. Leaves unbalanced negative charges, creating membrane potential. Forces all other K+ inside to counteract K+ flow out--here, E.G. for K+=0, even though higher K+ exists inside the cell than out. Resting membrane potential with Nernst equation expresses equilibrium--flow of negative and positive ions across the membrane is precisely balanced. (-20--200 mV (animal cells))-- reflection of E.G. of K+. When the cell is stimulated other ion channels in the plasma membrane open, changing permeability--whether ions enter/leave depends on the direction of the E.G. Membrane potential at any time depends on both state of ion channels and the concentration on either side of the plasma membrane. Rapid opening and closing of ion channels matters most in cell signaling. Measuring changes in electrical current is the main method to study ion movements and channels--proton-clamp recording provides direct picture of how ion channels behave. Fine glass tube is used as microelectrode to isolate and make electrical contact with a small area of the membrane at the surface of the cell. Technique makes it possible to record the activity of ion channels in all sorts of cell types--particularly large nerve/muscle cells. By varying concentrations in ions on either side of the patch, can test which ions will go through channels in the patch--voltage/membrane potential can be set and clamped at a value. Random thermal environments trigger on/off channels even though conditions are constant. “All or none” reaction for channels--fully open or closed. Ion channels differ with one another primarily with respect to ion selectivity and gating. For voltage-gated channels, the probability of having open controlled by membrane potential--for ligand- gated channel, controlled by binding of some molecule (ligand) to the channel. For mechanically-gated channels, opening is controlled by mechanical force applied to the channel. Auditory hair cells are a good example of cells that depend on mechanically gated channels. Vibrations pull channels open, flowing ions into hair cells--sets up electrical signal to auditory nerve to the brain. Voltage gated ion channels have domains of voltage sensors that are extremely sensitive to changes in membrane potential and changes above certain threshold value, exert sufficient electrical force on these domains to encourage the channel to switch from closed to open conformation (membrane potential change) alters probability that gate will open. When one type of voltage-gated ion channel opens, membrane potential can change, activate/inactivate other voltage channels--fundamental to electrical signaling. Neuron’s task is to receive, integrate, transmit signals and carry signals inward from sense organs such as eyes and ears to the central nervous system--brain and spinal cord. In CNS, neurons signal through networks. Every neuron has a cell body with a nucleus and long extensions radiating. Usually has one long extension--axon--which conducts electrical signals away from the cell body to distant target cells; shorter dendrites which radiate like antennae and provide larger surface area receive signals. Axon commonly divides at the far end into many branches, each which end in a nerve terminal so a message can be passed simultaneously to many target cells. Branches of dendrites can be extensive too. Neuron is stimulated by a signal delivered to localized site on the surface, initiated change in membrane potential--to spread, goes from dendrite to axon terminals to next dendrite, forming a neural circuit. Local change in membrane potential generated by a signal can be spread passively but rapidly becomes weaker with distance. Over short distances, unimportant, but for long-distance, pressure spread inadequate. Employ active signaling mechanism here, local electrical stimulus of sufficient strength triggers explosion of electrical activity in the plasma membrane that propagates constantly renewing--action potential or nerve impulse can carry long distance message. When neuron stimulated, membrane potential of the plasma membrane shifts to less negative value (towards zero). If depolarization sufficiently large, causes voltage-gated Na+ channels in the membrane to open transiently at the site. As these channels flicker open, allows a small amount of Na+ to enter the cell down a steep E.G. Influx of positive depolarizes further, opening more Na+ channels. Happens until the membrane potential is positive (+40 mV)--where electrochemical driving force for driving Na+ channels is zero--“timer” of Na+ channels causes them to inactivate even when the membrane is depolarized--remain inactivated until potential returns to initial negative value. During action potential, Na+ channels do not act alone--depolarized axonal membrane is helped to return to resting potential by opening voltage- gated K+ channels--open in response to depolarization, but not as fast a Na+ and stay open as long as the membrane is depolarized. When depolarization reaches peak, K+ ions start to flow out of the cell--brings back to resting state. Action potential spreads outwards as travelling wave from the initial side of depolarization, eventually reaching axon terminals. After action, Na+ pumps in the axon plasma to restore ion gradients of resting cell. 20% of the brain consumes the energy generated from metabolism of food to power the pump. When an action potential reaches nerve terminals at the end of an axon, the signal must be relayed to target cells that signal contact (muscle/nerve cells). Signal is transmitted to junction synapses. At most, the plasma of cells presynaptic (receiving)/postsynaptic (transmitting) the message are separated by the synaptic cleft, to which the electrical signal cannot cross. TO transmit, electrical signal is turned to chemical in the form of a neurotransmitter. Initially stored in the nerve terminals within membrane enclosed synaptic vesicles. When the action potential reaches the nerve terminal, some of the synaptic vesicles fuse with the plasma membrane, releasing neurotransmitters to the synaptic cleft--activates voltage-gated- Ca2+ channels, concentrated in the plasma membrane of the presynaptic nerve terminal. Because the concentration outside the terminal is 1000x greater than free Ca2+ in the cytosol, Ca2+ rushes in through the open channels--increase triggers membrane fusion that releases neurotransmitters--secreted into the synaptic cleft--binds to neurotransmitter receptors concentrated in the postsynaptic plasma membrane of the target cell--causes change in the membrane potential of the target cell which if big enough can fire action potential. Neurotransmitter is renewed from the synaptic cleft by enzymes/uptake into neighboring non-neural cells. Limits duration and spread of signal to quiet both pre/postsynaptic cell. Different types of neurotransmitters--rapid responses depend on receptors that are transmitter-gated ion channels--constitute a subclass of ligand-gated ion channels and function is to convert a chemical signal carried by neurotransmitters back into an electrical one. Channels open transiently in response to binding and changing ion permeability of postsynaptic membrane. Causes change in membrane potential. If the change is big enough, it will depolarize the post-synaptic cell and trigger an action potential. Good example is at the neuromuscular junction--special synapse is formed between the motor neuron and skeletal muscle cells-in vertebrae, the neurotransmitter is acetylcholine, and gate ion channel is acetyl receptor. But not all neurotransmitters excite the postsynaptic cell. Can either excite/inhibit, character of receptor that determines how the postsynaptic cell will respond. Receptors for excitatory neurotransmitters are ligand-gated cation channels-when bound, open for influx of Na+ which depolarizes the plasma membrane and activated. Inhibitor receptors for example: GABA and glycine are ligand-gated Cl- channels--increases permeability to Cl-, inhibits postsynaptic cell by making it harder to depolarize (curare (toxin)) causes muscle paralysis when bound to receptors--strychnine (rat poison) causes muscle spasms, death by blocking inhibitory glycine receptors on neurons in CNS. Many drugs for treatment for insomnia, anxiety, depression, schizophrenia act by binding to the transmitter-gated ion channels. Sedatives/tranquilizers affect by binding to GABA-gated Cl- channels--makes even more sensitive to GABA inhibition. Number of distinct types of neurotransmitter receptors that fall into a small number of families with subtle electrophysiological properties. For a signal to pass from one neuron to the next, nerve terminal of presynaptic cell converts an electrical signal to chemical, then diffusion across the synaptic cleft to the postsynaptic cell can convert to electric signal. Chemical synapses allow for combining signals, interpreting, recording, computing appropriate output from many inputs advanced by interplay of different types of ion channels in a neuron’s plasma membrane. In addition to integrating chemical inputs, can adjust the magnitude of the response--synaptic plasticity triggered by Ca2+ influx through special channel in the postsynaptic plasma membrane, which leads to functional alterations, number of neurotransmitters released, way postsynaptic cell responds or both--play important part in learning/memory. Photosynthetic algae use light gated channels to sense and go to sunlight. In response to blue light--channel rhodopsin (ex: aggressive mouse when expose to blue light in hypothalamus)--optogenetics. Advanced Cell Bio Chapter 14 Notes Part 1 It is thought that the earliest cells may have produced ATP by breaking down organic molecules generated by geochemical processes--fermentation occurs in present day cells, where they use energy from paerial oxidation of energy rich foods for ATP. Main chemical currency of energy is ATP< small amounts generated during glycolysis in the cytosol of all cells--but for most cells, ATP is produced by oxidative phosphorylation (O.P.). Generation of ATP by O.P. differs in the way ATP is produced during glycolysis in that it requires a membrane--in eukarya, takes place in mitochondria and depends on electron transport that moves H+ across the inner mitochondrial membrane. Consists of 2 stages, one set up E.G. proton gradient, other uses the gradient to generate ATP. Both are carried out by special protein complexes in the membrane. 1. In stage 1, high energy electrons derived from oxidation of food molecules, from sunlight or other sources, are transferred along a series of electron carriers--electron transport chain (E.T.C.)--embedded in the membrane. Electron transfers release energy used to pump protons, derived from H2O ubiquitous in cells, across membrane and thus generated an electron proton gradient. Ion gradient across a membrane is a form of stored energy to do work when ions are allowed to flow back down E.G. 2. Protons flow down E.G. through protein complex ATP synthase, which catalyzes the energy a synthesis of ATP from ADP and inorganic Phosphate (Pi). Ions like to bind, allowing proton gradient to drive ATP production. Together, these two stages are called chemiosmotic coupling Mechanism for making ATP arose very early in life’s history. Some type of ATP generating processes occur in the plasma membrane of modern bacteria and archaea. Process in eukaryotic mitochondria and chloroplasts evolved from engulfed bacterial cells more than 1 billion ya. Also harbor bacteria-like biosynthesis machinery to make RNA and protein and retain their own genomes. Many chloroplasts like cyanobacteria--photosynthetic bacteria from which chloroplasts are thought to have originated from. Although mitochondria/chloroplasts still have DNA, the bacteria that gave rise gave up many genes for independent living. The genes moved to the cell nucleus, however, and continue to carry out products of proteins that they import to carry out special functions, including the generation of ATP. Mitochondria present in nearly all eukaryotic cells, where they produce the bulk of the cell’s ATP. Without mitochondria, eukarya would rely on inefficient glycolysis. When glucose is converted to pyruvate by glycolysis in the cytosol, only 2 ATP/glucose procures, 10%< of free energy available. 30 molecules of ATP/glucose through mitochondria are produced. Patients with MERRF are deficient in multiple proteins required for electron transport, results in muscle weakness, heart problems, dementia. Muscle and nerve cells are especially sensitive to mitochondrial defects because of a need for ATP. Isolated mitochondria are generally similar in size and shape to bacterial ancestors. Although no longer capable of living independently, mitochondria are remarkably adaptable and can adjust to location, shape and number to suit the needs of the cell. Usually placed in an area of high energy consumption (ex: around sperm tail or contractile fiber). In other cells, fuse to form elongated, dynamic tubular networks, diffusely distributed though the cytosol--networks are dynamic, constantly breaking and fusion. Mitochondria are present in large numbers but vary in cell type and energy needs. Individual mitochondrion is bound by 2 highly specialized membranes--one surrounding the other. Outer and inner membranes create large internal space-- matrix--and narrower intermembrane space. When purified mitochondria are gently fractured into separate components and contents analyzed, each space has a unique collection of proteins. The outer membrane contains many molecules of transport protein porin which forms wide aqueous channels through the lipid bilayer. Center membrane like a sieve permeable to all molecules of 5000 daltons or less, including small proteins. Makes intermembrane space chemically equivalent to the cytosol with respect to small uncharged and inorganic ions it contains. Inner membrane is impermeable to the passage of ions and most small molecules, except where a path is provided by a specific membrane transport protein. Mitochondrial matrix only has molecules selectively transported into the matrix across the intermembrane and contents are highly specialized. The inner mitochondrial membrane is the site of O.P., has proteins of ETC, proton pumps and ATP synthase need for ATP production. Also has a variety of transport proteins that allow entry of selected small molecules--ex: pyruvate and fatty acids that will be oxidized by the mitochondria into the matrix. The inner membrane is highly convoluted forming cristae foldings that project into the matrix space. Greatly increases the surface area of the membrane. Generation of ATP powered by the flow of electrons is derived from the burn of carbs, fats, other fuels during glycolysis and the citric acid cycle. High energy electrons provided by activated carriers generated during the 2 stages of catabolism, with the majority churned out by the citric acid cycle that operates in the mitochondrial matrix. Citric acid cycled gets fuel needed to produce activated carriers from food molecules that make their way into the mitochondria by the cytosol. Both pyruvate from the glycolysis and fatty acids derived from the breakdown of fats can enter into the mitochondrial intermembrane space through porins of the outer membrane. Fuel molecules are then transported to the matrix, where they are converted to acetyl CoA--groups oxidized to CO2 via citric acid cycle. Some of the energy derived is saved in the form of high energy electrons, held by NADPH and FADH2--then donate electrons to the ETC in the inner membrane. Chemiosmotic generation of energy begins when activated NADH and FADH2 carriers donate high energy electrons to ETC in the inner membrane, oxidizing to NAD+ and FAD in the process. Electrons are quickly passed along chemically to molecular O2 to make H2O. Stepwise movement of electrons through the ETC releases energy that can be used to pump protons across the inner membrane. Resulting proton gradient is used to drive synthesis of ATP. Inner membrane serves as a device to convert energy in electrons of NADH/FADH2 into the P bond of ATP molecule--oxidative phosphorylation involves the consumption of O2 and the addition of P group to ADP to make ATP. Source of high energy electron differs between different organisms and processes. In cellular respiration, high electron ultimately derived from sugars or fats. In photosynthesis, high electrons come from chlorophyll which captures light energy. And many single celled organisms use inorganic substances to make ATP. ETC or respiratory chain has more than 40 proteins, grouped into 3 large respiratory enzyme complexes---contain multiple individual proteins, including transmembrane proteins that anchor the complex firmly in the inner mitochondrial membrane. 3 enzyme complexes in the order they receive electrons: NADH dehydrogenase complex, cytochrome c reductase complex, cytochrome c oxidase complex. Each contains metal ions and other chemical groups that acts as stepping stones to facilitate the passage of electrons. Movement accompanied by proton pumping form the mitochondrial matrix to the intermembrane space. Each can be though as a proton pump. First respiratory complex in chain, NADH accepts the electron from NADH< extracted in the form of H-, converted to proton and 2 high energy electrons catalyzed by NADH dehydrogenase. Electrons then passed along the chain to other complexes using mobile electron carries to ferry electrons between complexes. Transfer is energetically favorable. Final reaction is O2-requiring step and consumes almost all the O2 we breathe. Without a mechanism for harnessing energy released form the transfer of electron NADH to O2, energy would be released as heat. Cells recover by the energy that is used for proton pumping. Generates H+ (pH) gradient across the inner membrane. pH in the matrix is 7.9 and intermembrane is 7.2 ( same as cytosol). Proton umping generated by a membrane potential across the inner membrane; as H+ flows out, the matrix side becomes negative and intermembrane side is positive. Steep E.G. from H+ to flow into the matrix because of ion concentration and membrane potential--proton motive force--more membrane potential, more energy stored in the proton gradient. In most cells, H+ gradient is used to drive synthesis from ADP and Pi, Device that makes this possible is ATP synthase--large, multisubunit protein embedded in the inner membrane--same enzyme for all cells. Part of the protein that catalyzes phosphorylation of ADP is shape like a lollipop, projects into the mitochondrial matrix and is attached by a central stalk to H+ carrier and spins rapidly like a motor, rubs against the protons to alter the conformation and prompt to form ATP--3 molecules of ATP/revolution produced. ATP synthase can also operate in reverse-- using ATP hydrolysis to pump protons uphill against E.G., synthase operates like H+ pumps--can make/use ATP depending on the magnitude of E. proton G. across the membrane in which the enzyme is embedded, In bacteria that can grow areo/anaerobically, direction in which ATP synthase works routinely reversed when bacteria loses/runs out of O2. ATP synthase uses ATP from glycolysis to pump protons out to create proton gradient to import essential nutrients. Coupled transport across inner mitochondrial membrane also driven by E.P.G.--ex: pyruvate and Pi transported inward with protons as they move down EG to matrix. Other transporters take advantage of the membrane potential generated by EPG which makes the makes the matrix side of the inner membrane more negative than the intermembrane side. The antiport carrier protein exploits this voltage gradient to export ATP and bring ADP in. EPG drives the formation of ATP (-4) and transport of selected metabolites across the inner mitochondrial membrane. Due to nucleotide exchange, ADP (-3) from hydrolysis in the cytosol is randomly driven to the mitochondria for recharging, concentration of ATP in cytosol is kept about 10x higher than ADP--without activity of mitochondria, ATP levels would fall and cell would eventually die (cyanide blocks electron transportation in the inner mitochondria). Much of the energy carried by NADH/FADH2 ultimately converts into bond energy of ATP. How much ATP each of these activated carriers can produce depends on several factors, including where electrons enter the respiratory chain. NADH molecules produced in the mitochondria during the citric acid cycle pass high electrons to NADH hydrogenase complex--first complex. As electrons pass from one complex to the next, they promote proton pumping across the inner membrane. Each NADH=generated 2.5 ATP. FADH2 pass electrons to carrier ubiquinone-- promotes less pumping of protons, produces only 1.5 ATP. Almost 50% of total energy by fats/sugars stored in bonds of ATP during respiration. Transmembrane proton gradients drive the process of ETC ATP production (embedded in membranes). H2O serves as a ready reservoir for accepting/donating protons--these often accompany electrons transferred during oxidation (loses electron + H+)/reduction (gains electron + H+). ET pass spontaneously from a molecule with low affinity for outer shell electrons to higher. NADH with lower electron affinity, passes electron to NADH dehydrogenase complex. Redox pairs: ↔ NADH NAD+ +H+ + 2 electrons (tendency to donate/accept measured by redox potential) Electrons move spontaneously from redox pair with low redox potential (NADH/NAD+ (-320 mV/low)) or low affinity for electrons, to high, like O2/H2O, If Gibbs free energy is negative, it is spontaneous--transfer of electrons must be done in small steps to not cause extensive force and have all energy released as heat. When passing from one respiratory complex to the next, electrons formed by electron carries that diffuse freely within the lipid bilayer. In the mitochondrial respiratory chain, small ubiquinone picks up electrons from NADH complex and delivers to the cytochrome c complex. Quinone functions similarly during electron transport in photosynthesis. Ubiquinone can accept/donate 1 or 2 electrons, picks up 1 H+ from H2O with each electron it carries. Lies between both complexes in tendency to gain/lose electrons. Also serves as entry point for electrons donated by FADH2 generated during the citric acid cycle and from fatty acid oxidation. Redox potential of different level complexes influence where used along the ETC. Iron Sulfur centers have low affinities for electrons, prominent in electron carriers that operate in the early part of the chain. I.S. center in NADH complex passes electrons to ubiquinone. Later, iron atoms in heme groups to the cytochrome proteins common electron carriers. These heme groups give cytochromes; ex: cytochrome c reductase/oxidase their color. Cytochrome proteins increase the redox potential further down the mitochondrial ETC they are located. Cytochrome c accepts electrons from reductase complex--has redox potential between the cytochromes it interacts with. Advanced Cell Bio Chapter 14 part 2 Notes! Virtually all organic material in present cells is produced by photosynthesis--series of light driven reactions that creates organic molecules from atmospheric CO2. Plants, algae and photosynthetic bacteria use electrons from H2O and energy of sunlight to convert CO2 into organic compounds. In the course, H2O is split, releasing O2 which supports oxidative phosphorylation (O.P.) in animals, plants and aerobic bacteria. In plants, photosynthesis is carried out by the chloroplast--contains light capturing pigments such as the green pigment chlorophyll. In plants, leaves are major sites of photosynthesis--only occurs in the day--produces ATP and NADPH, Can be used to convert CO2 into sugar (carbon fixation). Chloroplasts are larger than mitochondria, both organized along structurally similar principles. Chloroplasts have a highly permeable outer membrane and less permeable inner membrane, in which many transport proteins are embedded. Together with the intermembrane space, they form the chloroplast envelope. Inner membrane surrounds the stroma--analogous to the mitochondrial matrix and has many metabolic enzymes. The inner membrane of the chloroplast does not have photosynthetic machinery--ETC, light-capturing, ATP synthase located in the thylakoid membrane. Folded to form disk-like sacs--thylakoids--arranged in stacks called grana. Space between other thylakoids connected--thylakoid space separated from stroma. Photosynthesis: light energy+ CO2 + H2O = sugars + O2 + heat energy st 1 stage-- equivalent to OP in mitochondria, ETC in thylakoid membrane harnesses the energy of electron transport to pump protons to space; gradient drives ATP synthase to synthesize high electrons donated to photosynthetic ETC come from 1 molecule of chlorophyll that absorbed energy from sunlight--light reactions. Electrons down the ETC are donated to NADP+ to make NADPH 2ndstage--ATP and NADPH from photosynthetic ETC are used to manufacture sugar from CO2 (carbon fixation). Can occur without sunlight--dark reactions. Begin in the stroma to generate 3 carbon sugar glyceraldehyde 3-phosphate-- exported to cytosol, where used to produce sucrose and other organic molecules. Stage 1 and 2 linked by feedback mechanisms. Most chloroplasts absorb at blue/red wavelength (430/660). Absorb green poorly. Electrons in chlorophyll distributed in decentralized cloud around the molecule light- absorbing porphyrin ring. When light hits the molecule, excites electrons in this network, perturbing electron distribution--high energy unstable, so chlorophyll converts energy. In thylakoid membrane of plants and plasma membrane of photosynthetic bacteria, chlorophyll are held in multiprotein complexes--photosystems. Each consists of antenna complexes which capture light energy and reaction center that converts it to chemical energy. In each antenna complex, hundreds of chlorophyll molecules are arranged so light energy captured by one chlorophyll can transfer to the next; energy jumps randomly-at some point the wandering energy will encounter a chlorophyll dimer--special pair--which holds electrons at a lower energy than others--becomes trapped. Part of the reaction center--transmembrane complex of proteins and pigments though to have first evolved more than 3 billion ya--within, special pair is positioned next to a set of electron carriers that accept high electrons from excited special pair. Converts light energy to chemical energy. As soon as electrons are handed off, the special pair becomes positively charged, electron carrier negatively. Rapid movement creates charge separation that sets in motion flow of electrons from the reaction center to an ETC. Photosynthesis is ultimately a biosynthetic process, and to build organic molecules from CO2, need huge input of energy in the form of ATP and reducing power (NADPH). To generate, use photosystems. Photosystem II absorbs light energy, reaction center passes electrons to mobile electron carrier (plastiquinone)--part of Proton ETC. Carrier transfers high energy electrons to proton pump, which uses the movement of electrons to generate electric proton gradient--drives production of ATP by ATP synthase in the thylakoid membrane. Photosystem I at the same time captures energy from sunlight--passes high electrons to different mobile electron carrier, which brings them to an enzyme that uses them to reduce NADP+ to NADPH--combined work produced used in Photosystem 2. When electron carrier takes from the chlorophyll special pair, electrons must be replaced--in photosystem II--replaced by special protein complex that removes electrons from water (water- splitting enzyme (has manganese--extracts H2O electrons one at a time-4 extracted--O2 released))--Photosystem I get electrons from Photosystem II--passed from proton pump to plastocyanin (electron carrier)--to Photosystem I. Movement of electrons from tight H2O to loose NADPH--enough energy left over to power ETC that links both Photosystems for ATP synthase. Light reactions of photosystem generates ATP and NADPH in the chloroplast stroma--but inner membrane impermeable to both--can’t be exported. Instead used to produce sugars via carbon fixation. CO2 from air is attached to 5-C sugar derivative, ribulose-1,5-biphosphate to make 2 3-phosphoglycerol. Catalyzed in stroma by enzyme Rubisco--more slow, 3 molecules of substrate processed/second-- surplus Rubisco used to keep up. Rubisco often represents more than 50% of total chloroplast protein. Although product of carbohydrates by CO2/H2O energetically unfavorable, fixation Rubisco energetically favorable because continuous supply of energy rich ribulose 1,5- biphosphate is fed into it. As the compound is consumed by the addition of Co2, it is replenished by ATP and NADPH from photosynthetic light reactions. In carbon fixation cycle, 9 ATP and 6 NADPH consumed. Glyceraldehyde 3-phosphate is the final product. If in excess, can be converted to start--stored in stroma. Can also be converted to fat. At night, can be broken down to sugars and fatty acids--some exported sugar enters glycolytic pathway to be converted to pyruvate, can enter plant cell mitochondria and be fed to the citric acid cycle--ultimately becoming leading to the production of ATP by O.P. Glyceraldehyde can also be used to make sucrose. O.P. evolved in stages. First, H+ pump, then ETC, EPG, Photosystem reaction centers, use of H2O, O2. Chemiosmotic coupling is an ancient process--lifestyle of Methanococcus--lives in thermal vents--uses nitrogen fixation. Advanced Cell Bio Chapter 15! To keep chemical reactions isolated, different strategies are used--ex: aggregating different enzyme required to catalyze a sequence of reactions into large nd multicomponent complexes--ex: for DNA/TNA/proteins synthesis. 2 strategy, highly developed in eukarya--confine different metabolic processes and proteins required within different membrane enclosed compartments. Eukarya elaborately subdivided by internal membranes--organelles contain unique set of molecules and carry out a specialized function. Membrane-enclosed organelles are surrounded by the cytosol (endorse by the plasma membrane. Nucleus generally the most prominent organelle in eukarya--surrounded by 2x membrane (nuclear envelope))--Communicates with cytosol via nuclear pores that perforate envelope/ Outer nuclear membrane continuous with the ER-system of interconnected sacs and tubes of membrane that extends through most of the cell--large areas of the ER (synthesizes new membranes) have ribosomes attached to the cytosolic surface and are rough ER. Ribosomes synthesize proteins delivered to ER interior (lumen)--smooth ER lacks ribosomes--scanty in most cells but highly developed in others (ex: site for steroid hormone synthesis/alcohol detoxification). Can also sequester Ca2+ from the cytosol. Golgi near the nucleus receives protons/lipids from the ER, modifies, dispatches. Small sacs of lysosomes degrade worn organelles and macromolecules and particles by endocytosis. Endocytosed materials first go through endosomes which sort molecules and recycle some to the plasma membrane. Peroxisomes are small organelles that have enzymes used in oxidative reactions that break down lipids//toxic molecules. Mitochondria and chloroplast as surrounded by a double membrane and sites of oxidative phosphorylation and photosynthesis--both contain internal membranes highly specialized for the production of ATP. Most membrane enclosed organelles are held in relative locations by attachment to the cytoskeleton, especially microtubules. Cytoskeletal filaments provide tracks for moving organelles around and directing traffic of vesicles between one organelle and another--driven by motor proteins using ATP hydrolysis energy--membrane enclosed organelles compose ½ of the volume of the cell; in terns if mass/area, the plasma membrane is minor to others. Compartments in eukarya probably evolved in stages. Precursors to the first eukaryotic cells are thought to be simple microorganisms resembling bacteria, with a plasma membrane but no internal membranes. The plasma membrane would have performed all cell-dependent functions, including ATP synthesis, lipid synthesis, like most bacteria--not possible in eukarya. 2 ways membrane enclosed organelles could have arisen--nucleus, ER/Golgi, lysosomes most likely from the plasma membrane--endomembrane system. Mitochondria and chloroplast s are thought to have originated via bacteria engulfed by eukarya--still isolated from veriscular traffic that corrects interiors of the rest of other membrane enclosed organelles--possess their own small genomes and can make some of their own proteins. As cells grow, membrane enclosed organelles enlarge by the incorporation of new molecules; then divide and are distributed between 2 daughter cells. Organelles growth requires the supply of new lipids to make more membrane supply of appropriate proteins--new and soluble that will occupy the interior of the organelle. Even in a cell that’s not dividing proteins are made continually--must be made accurately to be delivered to the appropriate organelle for secretion and some to replace the organelle proteins that have been degraded. Directing newly made proteins to correct organelle necessary for any cell to grow and divide or just function properly. For some,(mitochondria, chloroplasts, peroxisomes, interior nucleus) proteins are delivered directly from the cytoplasm to the Golgi, lysosomes, endosomes, inner nucleus, proteins and lipids delivered indirectly via ER. Proteins enter the ER via the cytosol, some retained, must be transported by vesicles to the Golgi and onward. Peroxisomes acquire some membrane proteins from the ER, the bulk from the cytosol. Synthesis of virtually all proteins in the cell begins on ribosomes in the cytosol--exceptions are from the mitochondria/chloroplast proteins made inside--most still from the cytosol. Fate depends on the amino acid sequence, which can have sorting signal that directs the protein to the organelle--proteins lacking remain permanently in the cytosol. When a membrane-enclosed organelle imports H2O soluble proteins it faces a problem--how to transport across the membrane: 1. Protein to nucleus goes via nuclear pores (penetrate inner and outer membranes) 2. Protein to ER, mitochondria and chloroplast are through protein translocators in the membrane. The protein must usually unfold to snake across the membrane through the translocator. Bacteria have a similar protein translocator in the plasma membrane 3. Protein from the ER on is transported by transport vesicles, which pinch off from membranes of one compartment of the endomembrane system to fuse with another. In the process, soluble cargo proteins are delivered as well as prteins and lipids that are part of the vesicle membrane. Typical sorting signal on a protein: continuous stretch of amino acid sequence--15- 60 acids long. Signal sequence is often (not always) removed from the finished protein once sorted. Signal sequences are necessary and sufficient to direct proteins to a particular destination. Deleting a sequence from the ER converts it into a cytosolic protein and vice versa. Signal sequences specifying the same destination can vary greatly even with the same function: physical properties such as hydrophobicity or placement of charged amino acids often appear to be more important for function of signals than the exact amino acid sequence. The nuclear envelope encloses nuclear DNA and defines the nuclear compartment-- forms from 2 concentric membranes--inner nuclear membrane contains some proteins that act as binding sites for chromosomes and others that anchor the nuclear lamina--finely woven meshwork of protein filaments that lines the inner face of the membrane and gives structural support for the nuclear envelope. Composition of the outer nuclear membrane closely resembles the membrane of the ER--continuous. Nuclear envelope in eukarya is perforated by nuclear pores that form gates where molecules enter or leave--large complex of 30 different proteins-- contain many unstructured regions in which polypeptide chains are disordered. Creates tangled meshwork. To enter the pore, molecules must have appropriate sorting signal--nuclear localization signal--1 or 2 sequences with positively charged arginines/lysines (basic). NLS recognized by the cytosolic proteins--nuclear import receptors. Help direct newly made protein to the nuclear pore by interacting with tentacle-like fibrils from rim of pore. Once there, NIR (nuclear import receptors) grab onto short repeated amino sequences within the tangle of pore proteins at the center--when empty, the sequences bind to each other in loosely packed gel. NIR open passage through the meshwork--bump into one repeat sequence to another until they reach the nucleus. NIR then returns to the cytosol to be reused. Energy for transport is provided by GTP mediated by GTPase Ran--operation of pore runs at good speed. Pores transport proteins in fully folded conformations and ribosome components as assembled particles--different from other organelles. Both mitochondria and chloroplast are surrounded by inner/outer membranes, both organelles specialize in ATP generation. Most mitochondria/chloroplast proteins are encoded by genes in the nucleus and are imported from the cytosol. Usually have a signal sequence at the N-terminus--proteins destined for either organelle are translocated simultaneously across both inner and outer membranes at special sites where the 2 membranes contact each other. Each protein is unfolded as it is transported--signal sequence is removed after complete. Chaperone proteins inside organelles help pull the protons across both membranes and fold once inside. For protein to get to specific site within the organelle, it usually requires further sorting signals--exposed after the first signal sequence is removed. Insertion of transmembrane proteins across the inner membrane, for example, is guided by signal sequences. Growth and maintenance of mitochondria and chloroplast require the import of new proteins and incorporation of new lipids into the membranes of the organelles. Most membrane phospholipids are thought to be imported form the ER--transported by lipid carrying proteins that extract phosphorous molecule from one membrane and delivered into another. Peroxisomes generally contain one or more enzymes that produce Hydrogen peroxide; present in all eukarya, break down a variety of molecules including toxins, alcohols, fatty acids. Also synthesizes certain phospholipids including those abundant in myelin sheath that insulates nerve cell axons. Peroxisomes acquire most proteins via selective transport from the cytosol--short sequences of 3 amino acids that serves as an import signal--recognized by the receptor proteins in the cytosol, one which escorts cargo protein into the peroxisome before returning to the cytosol. Peroxisome membrane has a protein translocator that aids in transport--no need to unfold for import in to the peroxisome. A few membrane proteins for the peroxisome arrive via vesicles for the ER--fuse with preexisting peroxisomes or import peroxisome proteins from the cytosol to grow into mature peroxisome (disease of Zellweger block peroxisome protein import--most die within 6 mo.) The ER is the most extensive membrane system in the cell. Serves as entry point for proteins destined for other organelles as well as for the ER itself. Proteins for the Golgi, endosomes, lysosomes and cell surface, all enter the ER from the cytosol. Once inside the lumen or embedded in the ER membrane, individual proteins will not reenter the cytosol during onward journey. Will be ferried by transport vesicles from cytosol to ER--1. H2O-soluble--completely translocated across the ER membrane and released to lumen, 2. Prospective transmembrane proteins-only partly translocated across the ER and become embedded. H2O soluble are destined for secretion by release at the cell surface or lumen of an organelle in the endomembrane system. Transmembrane proteins are destined to reside in the membrane of one of these or plasma membrane. All initially directed by ER signal sequence, 8 or more hydrophobic amino acids are also involved in the translocation of the polypeptide chain completely synthesized--requires that the ribosome synthesizing be attached to the ER--these ribosomes dot the surface, creating rough ER. 2 separate populations of ribosomes--membrane-bound attached to cytosol side of the ER (and outer nuclear membrane)--making proteins to be translocated into the ER. Free ribosomes are unattached, make all other proteins encoded by nuclear DNA. Structurally functionally identical, differ in the products they make at any given time. When free ribosomes are making an ER protein., directs ribosome to ER. When mRNA with code for ER is being translated, helps attach the poly ribosome to the ER. 2 protein components help guide ER signal sequence to ER membrane 1. Signal recognition particles (SRP) in cytosol, binds to ribosome and ER signal sequence when it emerges from the ribosome 2. SRP receptor, embedded in the ER, recognizes SRP. Binding of SRP to the ribosome with ER signal slows protein synthesis by that ribosome until the SRP engages with the receptor on the ER. Once bound, SRP is released. Receptor passes ribosome to protein translocator in the ER and protein synthesis recommences. The polypeptide is then threaded across the ER through a channel in the translocator. SRP and receptor molecule are matchmakers. ER signal functions to open the channel in the translocator, sequence remains bound to the channel while the rest of the polypeptide is threaded through as a large loop. Removed by transmembrane signal peptidase with active site facing the luminal side of the ER--cleaved signal is released into the lipid bilayer and degraded. Once C-terminus of soluble protein is passed through the translocation channel. The protein will be released into the ER lumen. Not all proteins by ER-bound ribosomes are release to the lumen, some are stuck as transmembrane proteins. Begins as translocation at the N-terminal sequence, but halted by a sequence of hydrophobic amino acids (stop-transfer sequence) further along the polypeptide chain. At this point, the translocation channel releases the growing polypeptide sideways into the lipid bilayer. N-terminal cleaved, stop- transfer stays in the bilayer, forming alpha-helical membrane spanning segment that anchors the protein in the membrane--ends as a single pass with N-terminus on lumen side, C-terminus on cytosol side--never changes orientation. In some transmembrane proteins, internal signal sequence is used to start protein transfer (start-transfer sequence). Never removed from polypeptide. Arrangement occurs in the same transmembrane proteins in which the polypeptide chain passes back and forth across the lipid bilayer--hydrophobic signal sequences thought to work in pairs and internal start transfer serves to initiate translocation until stop transfer reached, 2 hydrophobic sequences are then released into the bilayer to form alpha helices-- multipass. Down the polypeptide one sequence reinitiates translocation and causes the polypeptide to release, and so on for subsequent starts and stops--multipass membrane proteins stitches in bilayer as they are synthesized. Destination generally from ER lumen/membrane is Golgi--proteins/lipids modified and sorted. Vesicular transport extends outward from the ER to the plasma membrane and inward from the plasma membrane to lysosomes--as proteins are moved, they undergochemical modifications. Vesicular transport between membrane-enclosed compartments of the endomembrane system is highly organized. Major outward secretory pathway starts with the synthesis of proteins on the Er. Entry into the ER, leads though Golgi to cell surface--at the side brance, heads though the endosomes to lysosomes. Major inward endocytic pathway-- responsible for ingestion/degradation of extracellular molecules, moves materials from the plasma membrane through the endosomes to the lysosomes. Vescicles that bud form the membranes have a distinctive protein coat the cytosolic surface-- coated vesicles. After budding from the parent organelle, sheds coat to interact with the fusing membrane. Protein coat helps shape the membrane into bud an captures molecules for onward transport. Best studied vesicles have clathrin coat--but from Golgi to the plasmam membrane and from the plasma membrane to the endocytic pathway. At the plasma membrane, starts as clathrin-coated pit--basketike network on the cytosolic surface of the membrane. GTP binding protein dynamin assembles as a ring around the neck of the invaginating coated-pit--causes ring to constrict and pinch off the membrane with the help other proteins. Adaptins (coat proteins) secure clathrin to the vesicle membrane and help select cargo for transport. Molecules for onward transport have transport signals recognized by cargo receptors in the Golgi or plasma membrane. Adaptins help capture the specific cargo molecules by capturing the receptors--incorporate into the vesicle lumen. Adaptins on each vesicle different at each membrane. COP (coat-protein)-coated vesicles transport molecules between the ER and Golgi and from one part of the Golgi to another. Often, the vesicle is actively transported by motor proteins that move along the cytoskeletal fibers. Once a vesicle reaches its target, it recognizes and binds with the specific organelle by molecular markers--recognized by complimentary receptors. ID process depends on a diverse family of monomeric GTPases--Rab proteins--recognized by tethering proteins on the cytosolic surface of the target membrane. Each organelle and type of vesicle has a unique combination of Rab proteins. Additional recognition by transmembrane proteins is done by SNAREs, once tethering protein has captured the vesicle by grabbing Rab, v-SNAREs interact with t-SNAREs to firmly dock. SNAREs from docking also help the vesicle fuse by catalysis--delivers soluble contents and adds the membrane of the vesicle to the membrane of the organelle. For fusion, the bilayer of both membranes must come within 1.5 nm close to each other for lipids to intermix--water must be displaced from hydrophilic surfaces of the membranes--highly unfavorable--prevents random fusing. Vesicular traffic is not confined to the interior of the cell--exocytosis. Most proteins that enter the ER are chemically modified there. S-S bonds formed by oxidation of pairs of cysteine side domains is catalyzed by an enzyme that resides in the ER lumen. S-S help stabilize the structure of proteins that encounter degradation enzymes and pH changes-- after secretion or after plasma membrane incorporation. S-S bonds don’t form in the cytosol because the environment is reducing. Many proteins to the ER lumen/membrane are converted to glycoproteins in the ER by covalent attachment of short branched oligosaccharide side chains of X sugars--glycosylation is carried out by glycosylic enzymes in the ER but not in the cytosol. Oligosaccharides can protect the protein from degradation, hold in ER until folded well, help for packing as a transporter signal. In the ER, preformed oligosaccharide with 14 sugars attached en bloc to all proteins for glycosylation--oligosaccharide originally attached to specialized lipid (dolichol) in ER membrane, then transferred to an amino group of asparagine side chain immediately after the target emerges after translocation. In one step, catalyzed by oligosaccharyl transferase with active site exposed to the lumen side of the ER. Oligosaccharide with NH2 asparagine group called N-linked. ER retention signal is on C-terminal of the protein, recognized by membrane-bond receptor proteins in the ER and Golgi. Exit from ER is selective--if not folded correctly, proteins are stuck by being bound to chaperones--if still not folded well they are spat out into the cytosol for degradation. Ex: antibodies in the ER have 4 polypeptide chains--deleterious forms can occur--cystic fibrosis--transport protein is retained in the ER. Quality control in the ER can be overwhelmed--when that happens, misfolded proteins accumulate in the ER. If the buildup is large, it triggers complex program. Unfolded protein response (UPR) prompts cell to produce more ER--including chaperones and other proteins for quality control--allows cell to adjust to size or ER according to the load of proteins entering the secretory pathway. In some cases--cannot help. Self-destructs in apoptosis--may occur in adult onset diabetes. Golgi is usually near the nucleus, in animal cells close to the centrosome (cytoskeletal structure near the cell center). Consists of a collection of flattened non-enclosed sacs--cisternae--piled like pita bread (3-20 cis). Number of stacks varies--some cells have 1 large stack, others many small ones. Each stack has 2 face--entry-cis(adjacent to ER), exit-trans(found near plasma membrane). Outermost cisterna at each face connection to network of intermembraneous tubes and vesicles. Soluble proteins and membrane enter cis Golgi network via transport vesicles from the ER. Proteins travel via vesicles through cisternae in sequence by fusing one to the next. Proteins exit via trans network in vesicles for cell surface or another organelle in the endomembrane system. Many oligosaccharide chains added to the ER undergo modifications in the Golgi. In all eukarya, steady stream of vesicles buds from trans network and fuses with the plasma membrane via exocytosis. Constitutive exocytosis pathway supplies the plasma membrane with new lipids/proteins, enabling the plasma membrane to expand prior to cell division and refreshed old lipids/proteins in non-proliferating cells. Constitutive exocytosis pathway carries soluble proteins to the cell surface to be released to the outside by secretion. Some proteins remain attached to the cell surface; some incorporate to the extracellular surface--some diffuse to extracellular fluid to nourish/signal other cells. Entry to constitutive pathway does not require particular signal sequence. Regulatory exocytosis pathway--operates in cells specialized for secretion. Each specialized secretory cell produces large vesicles for later release--but off from trans Golgi and accumulates near the plasma membrane--wait for signal to fuse with the plasma and release contents. Proteins for regulatory secretion is sorted/packed in trans Golgi--those that travel this way have special surface properties that cause aggregation in ionic conditions (acid pH and higher Ca2+) that prevail in trans--pack into secretory vesicles and wait for signal. Proteins by constitutive pathway do not aggregate, carried automatically to the plasma membrane. Selective aggregation allows secretory proteins to be packed into vesicles at a higher conce


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