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Week 5 Lecture Notes

by: Luke Holden

Week 5 Lecture Notes BIOL 4610

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These notes cover the last bit of the secretory pathway and the endomembrane system. It contains personal notes as well as power point notes with helpful figures from her power point as well as fro...
Cell Biology
Susan Chapman
Class Notes
endomembrane system, secretory pathways
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This 13 page Class Notes was uploaded by Luke Holden on Friday October 7, 2016. The Class Notes belongs to BIOL 4610 at Clemson University taught by Susan Chapman in Fall 2016. Since its upload, it has received 20 views.


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Date Created: 10/07/16
Notes for Week 5 (9/22-9/27) Big Picture: We are marching our way through the endomembrane system and we are going to begin this week by talking about endosomes and then finish through peroxisomes. Endosomes Functions of Endosomes:  Endocytosis- bring material into the cell o Acquire things o Phagocytosis: eat solids o Pinocytosis: liquids are taken up o Process:  A small segment of the plasma membrane folds inward  Then it pinches off to form an endocytic vesicle containing ingested substances or particles o Receptor Mediated Endocytosis (Clathrin Dependent Endocytosis)-  This is a way to regulate how material comes into the cell by endocytosis  This is how you get cholesterol into the cell  Process  Specific molecules (ligands) bind to their receptors on the outer surface of the cell  Receptor-ligand complexes diffuse laterally and encounter specialized regions called coated pits, sites for collection and internalization of these complexes  These proteins—adaptor protein, clathrin, dynamin— induce curvature and invagination of the pit  Eventually, the pit pinches off forming a coated vesicle  The clathrin coat is released, leaving an uncoated vesicle  Coat proteins and dynamin are recycled to the plasma membrane, and the uncoated vesicle fuses with an early endosome  It can then go 3 ways from this point o Some complexes are carried to a lysosome for degradation o Some complexes are carried to the TGN, where they enter a variety of pathways o Complexes can also travel by transport vesicles to a different region of the plasma membrane, where they are secreted (transcytosis) This is such a helpful picture in regards to Receptor Mediated Endocytosis!!!! Know it well!  Low Density Lipoproteins-  When you carry these in you are bringing in cholesterol  Bring in too much LDL then you will have plaque build up  High in the liver  Some Variations of Receptor Mediated Endocytosis:  Epidermal Growth Factor o This is a type of stimulant that initiates cell growth o However, you can think of this as a negative feedback process o When EGF is endocytosed, the sensitivity to EGF is decreased because the amount of receptors has decreased o Therefore the process of cell division is controlled because as the amount of EGF increased the sensitivity decreases (desensitization)  If there is a mutation where too many receptors are on the membrane, then the cell can get overstimulated to which you will have unregulated cell growth (she might ask a question about this on the exam: (“if the EGF were to have a mutation….”)  As the endosome matures it becomes more acidic: this is due to the proton pumps on the membranes.  This is to recycle the receptors because they will dissociate from the membrane because it is too acidic. o Clathrin Independent Endocytosis  Very simple  constant rate  Are routed to an early endosome  Fluid-phase endocytosis is a type of pinocytosis for nonspecific internalization of extracellular fluid  It does not concentrate the ingested material  Exocytosis- push material out of the cell o Process of Exocytosis  Vesicles containing products for secretion move to the cell surface  The membrane of the vesicle fuses with the plasma membrane  Fusion with the plasma membrane discharges the contents of the vesicle  The membrane of the vesicle becomes part of the cell membrane o Glycoproteins or glycolipids that are attached to the inside of the vesicle will now appear on the outside of the cell. This is how to “tag” your cells which allows them to be specified from other organ cells  Secretory granules and vesicles: These are a part of the secretory pathway and these bud from the Golgi and are carried to the plasma membrane and undergo exocytosis o 3 types of secretion:  Constitutive Secretion  This is a type where this occurs all the time around the clock  Lipids, hormones, mucosa layer line the stomach in the intestinal lining undergo this process  Unregulated  Regulated Secretion  This is a controlled process where the vesicles build up in the cell  Requires a specific signal to active it  Receptor is on the outside of the cell uses secondary messenger to call for exocytosis  Regulatory vesicles form by budding from the TGN as immature secretory vesicles  It must undergo maturation process called condensation (this is just where they gang up with all of their buddies and condense) o Might undergo proteolytic processing  They will stay here until they get the signal to where they will fuse with the plasma membrane  Polarized Secretion (specific exits)  This is a really cool secretion to where it can release molecules to only one side of the cell  Intestinal cells are a great example Polarized Secretion Picture More about the Coated Vesicles:  Type of coat helps determine their destination  Help form the vesicle and increase the specificity of the vesicle  Several types: Coated Vesicile Coat Proteins origin Destination Clathrin Clathrin AP1, ARF TGN Endosomes Clathrin Clathrin AP2 Plasma membrane Endosomes COPI COP1, ARF Golgi Apperatus ER to golgi apparatus COPII COPII (Sec 13/31 EER Golgi Apperatus and Sec 23/24) and Sar 1 Caveolin Caveolin Plasma Membrane ER? o  Clathrin (Soccer Ball) o Surrounded by multimeric proteins such as clathrin and adaptor protein o The basic unit of clathrin lattices is a triskelion o So the math of the vesicle  3 Heavy Chains of Clathrin + 3 Light Chains of Clathrin = triskelion  two of adaptin + one medium chain + and one small chain= AP (These bind to the ends of the triskelion.  o They alternate hexagon and pentagon shapes to provide stability o Functions of forming the clathrin coat  provide some of the driving force for vesicle formation  allows the new coat to curve around the budding vesicle  Dynamin o This is a protein that is responsible for for closing off the vesicle o Hydrolyses GTP to constrict and cut off the plasma membrane from the vesicle  Uncoating the Vesicle o Requires ATP o Done by an un-coating ATPase  Another word about Clathrin o When conditions are acidic and Ca is present, Clathrin can form clathrin cages  COP 1 and 2 go between the ER and the Golgi Complex o COP1 is involved with Retrograde o Vesicles are coated with COP1 and ARF (ADP ribosylation factor) o ARF mediates COP 1 assembly  In the cytosol, ARF exists as part of an ARF-GDP complex  Upon meeting a guanine nucleotide exchange factor associated with the membrane, the GDP is exchanged for GTP  The resulting conformational change in ARF attaches it to the lipid bilayer  Once firmly anchored, ARF binds COPI multimeres  Assembly of the coat drives vesicle formation  Once the vesicle is formed, a protein in the donor membrane triggers hydrolysis of GTP to GDP, a conformational change in ARF, and release of the coat o COP 2 o Anterograde transport o In yeast, the COPII coat is assembled from two protein complexes (Sec 13/31 and Sec 23/24) and a small GTP-binding protein called Sar 1 o SarI is similar to ARF, and the process of coat formation is similar to COPI-coated vesicles o o Snare Hypothesis o This describes the specificty of the vesicles o Mediate Fusion Between Vesicles and Target Membranes o N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs)  Don’t have to know the name just the abbreviation.  These are proteins necessary for secretion o Proteins required for the hypothesis:  v-SNAREs (vesicle-SNAP receptors) are found on vesicles  t-SNAREs (target-SNAP receptors) are found on target membranes  They are complementary molecules o These are like little sensors that go before the vesicle to see where to bind to. o When the proteins reach their destination, proteins called Rab GTPases come and bind the t snare to the v snare by hydrolysis of ATP o When the vesical is fused NSF and a group of SNAPs (they are non specific) mediate release of the SNAREs of the donor and target membranes Lysosomes Big Picture: we are now on the final step of the secretory pathway and we are talking about the second last endomembrane type Lysosomes Lysosomes Characteristics  Contains Several digestive enzymes  Capable of degrading all major class of molecules  Lysosomes contain acid phosphatase and several other hydrolytic enzymes  They vary in size and shape but are usually about 0.5 µm in diameter, bounded by a single membrane  The luminal side of the membrane is coated with glycoproteins to protect the membrane from degradation  Lysosomes eventurally fill up with waste which is a factor of aging  Highly acidic on the inside o 4-5 pH ONLY WORKS IF THE pH REMAINS WITH IN THESE BOUNDRIES THUS WE NEED A PROTON PUMP o Proton pumps are responsible (ATP-Dependent Pumps) o Acid Hydrollases (All of the enzymes in the lysosome fall under this category) o Lysosomal enzymes are synthesized by ribosomes on rough ER and translocated inside o Lysosomal enzymes are delivered from the TGN to endosomes in transport vesicles o Over time, endosomes mature into late endosomes, with all the enzymes, but not engaged in digestion  Endosome development o Early lysosome 6-5.5 (1) o loses ability to fuse with endocytic vesicles (2) o The late endosome is packaged with material to be digested and newly synthesized digestive enzymes (3)  This protects the cell from hydrolytic enzymes o The last step in development of a lysosome is activation of the acid hydrolases (4)  This occurs as the internal environment becomes more acidic (pH 4.0–5.0)  This occurs through the pumping of protons or through fusion with an existing lysosome  2 Types of Digestion o Heterophagic Lysosomes (Nutrient containing)  Lysosomes containing substances that originated outside the cell are called  Phagocytic vacuoles become lysosomes by fusion with endosomes  But vesicles contain contents that was brought in by receptor- mediated endocytosis fuse with vesicles of the TGN containing acid hydrolases  Once digestion is complete, only the indigestible material remains in the lysosome in which it is now called a residual body  Some cells release contents by endocytosis others accumulate which contribute to cellular aging o Autophagic lysosomes (Waste Containing)  lysosomes with material on the inside of the cell  Cellular structures what needed to be broken down use this type of digestion  Macrophagy: this is for double membrane bound organelles that originate from the ER and form what is called autophagic vacuole (autophagosome)  Microphagy: a much smaller vacuole is formed which is surrounded by a single membrane layer  Defective in this process could be cancerous  Knocking out a gene in a mice that affect the autophagic process left the mice with a buildup of cellular waste and lung / breast tumors  o Extracellular Digestion  This is when lysosomes dump their enzymes on the outside of the cell to degrade something  The sperm doing this to degrade the protective barriers of an egg is a good example. o Lysosomal Storage Diseases  Over 40 found  due to the lack of a specific enzyme  enzyme replacement therapy- this the treatment of replacing a certain enzyme to help degrade the cellular material. Peroxisomes Characteristics  Not part of the endomembrane system o AKA not derived from the ER  Defining characteristic is the presence of catalase regarding H2O2  Animals have a crystalline core, consisting of crystalline urate oxidase  Plants have crystalline catalase  W/O this core it is hard to see microscopically Functions:  Linked to H2O2 metabolisms o Detoxification of harmful compounds o Oxidation of fatty acids o Metabolism of nitrogen-containing compounds o Catabolism of unusual substances A word about H2O2 metabolism  Oxidase generates H2O2 in peroxisomes  o The H2O2 is detoxified by catalase  It can go backwards to and function as a peroxidase o  THE RESULT IS THE SAME BECAUSE H2O2 IS DEGRADED!!!!! ROS (Reactive Oxygen Species)  Peroxisomes detoxify reactive oxygen species (H2O2, O2–)  If these accumulate, they can cause oxidative stress  Peroxisomal enzymes such as superoxide dismutase and others detoxify reactive oxygen species Peroxisome Biogenesis  forms from other peroxisomes or the fusion of vesicles  Peroxisomes increase in number as cells grow and divides- biogenesis  peroxins- These are proteins required for this process  Process: o Membrane components, matrix enzymes, and cofactors from the cytosol are incorporated into peroxisomes o Then, new peroxisomes are formed by division of a preexisting one o Some peroxisomes may obtain materials or form de novo from vesicles derived from the ER Protein Targeting to Peroxisome  Matrix Protiens o All proteins are pre-folded when they enter the peroxisome o they are nuclear encoded o 2Sequenes:  PTS 1  Most Common  Located on C Terminus  SKL (or -AKL, -ARL, or similar)  PTS 2  A few proteins  N-Terminus  -(R,K) – (L,V,I) – X5 – (H,Q) – (L,A,F) o Bringing it in  PEX 5 binds to PTS1  PEX 5 Binds to PEX13/14 receptor  PEX 5 Escorts the protein in  PEX 5 is exported back out by PEX 2 PEX 10 PEX 12  Matrix proteins are targeted independent of membrane proteins o Different targeting sequence of membrane proteins  PEX 19 is the likely receptor for this guy Lysosomal Disorders  A large number of disorders arise from defects in peroxisomal proteins  The most common is X-linked adrenoleukodystrophy  The defective protein in this case may be responsible for transporting long-chain fatty acids into the peroxisome for degradation


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