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What are the three kinds of protein coats?
- 1. COPII - anterograde transport from the ER
- 2. COPI - retrograde transport from the Golgi and the ERGIC
- 3. Clathrin - transport from plasma membrane and endosomes
What are the functions of protein coats?
- large protein cages that are spherical in shape
- 1) recruit cargo
- 2) drive vesicle formation - requires energy
Describe the seven steps of vesicle targeting and fusion.
- 1. Initiation and Cargo Selection - recruitment and activation of GTPase to membrane recruits coat proteins and associated cargo to budding site; GTPase is activated when it binds GTP
- 2. Budding - coat assembles into spherical cage driving vesicle formation
- 3. Scission - vesicle is released
- 4. Uncoating - hydrolysis of GTP releases coat - recycled for additional rounds of budding
- 5. Tethering - "long range interactions" tethering proteins (Rab GTPases and other tethering factors) target vesicles to correct target membrane
- 6. Docking - "short range interactions" SNAREs on both vesicle and target membranes engage and drive fusion
- 7. Fusion - the actual fusing of lipid bilayers between vesicle and target
What is the difference between a v-SNARE and a t-SNARE?
- A SNARE is an alpha helical membrane protein that interacts to drive membrane fusion.
- v-SNAREs are on the vesicle membrane while t-SNAREs are on the target membrane
Explain how fusion of the vesicle and target membranes is accomplished using SNAREs.
One v-SNARE interacts with with three t-SNAREs to form a trans-SNARE complex (two sets of complexes per vesicle). They interact to form a coiled-coil structure which brings the vesicle very close to target membrane. After fusion, the trans-SNARE complexes are now cis-SNARE complexes because all the proteins are on the same membrane. The cis-SNARE complexes must be unwound to recycle the proteins- requires GTP.
What are the two kinds of membrane traffic?
- 1) Endocytosis- process of removing membranous material from the PM via vesicle budding
- 2) Exocytosis - process of adding membranous material to the PM through vesicle fusion
What are the three kinds of endocytosis?
- 1) Pinocytosis - fluid, uses small vesicles, accounts for most of the endocytosis in most cells
- 2) Phagocytosis - large particles ie. macrophages ingesting bacteria
- 3) Receptor-Mediated - cell signaling, clathrin coat-mediated
Name the 8 steps of trafficking/sorting to the lysosome.
- 1) Lysosomal enzyme is phosphorylated on mannose in cis Golgi
- 2) M6P receptor binds enzyme and recruits clathrin
- 3) Enzyme receptor complex buds from Golgi
- 4) Vesicle uncoats
- 5) Vesicle fuses with the late endosome (pH 5)
- 6) Acidic pH of late endosome releases enzyme from M6P receptor
- 7) M6P receptor recycles back to trans Golgi
- 8) Enzyme is dephosphorylated and traffics to lysosome
Explain the 8 steps of receptor-mediated endocytosis.
- 1) Secreted lysosomal enzyme binds M6P receptor on PM
- 2) Enzyme receptor complex recruits clathrin and buds into cell
- 3) Vesicle uncoats and fuses with early endosome (pH 6)
- 4) Early endosome fuses with the late endosome (pH 5.5)
- 5) Acidic pH of late endosome releases enzyme from M6P receptor
- 6) M6P receptor recycles back to trans-Golgi or (7) PM
- 8) Enzyme is dephosphorylated and traffics to lysosome
What are the main functions of the lysosome?
- Vital to breaking down/recycling cellular components
- Acidic pH=5
- Receive hydrolytic enzymes from Golgi
- Receive material to degrade from endocytic pathway, phagocytosis, and autophagy
What is a residual body?
- material that can't be completely digested by lysosomes, occurs in long living cells
- called lipofuscin in neurons
- cells can exocytose the whole lysosome to get rid of the residual bodies
Provide two examples of the clinical relevance of lysosomes.
- Tay-Sachs Disease- hexosaminidase A (Hex-A) enzyme deficient
- lipid ganglioside GM2 accumulates abnormally in cells, especially in in the nerve cells of the brain
- Xanthomas- deposits of cholesterol in skin in patients with LDL receptor deficiency
- LDL receptor is not trafficked to PM or endocytosed properly; cells can't uptake LDL and therefore can't catabolize cholesterol
What are the two methods to traffic lipids?
- 1) Vesicular transport
- 2)hidden within lipid transfer proteins (LTP's) = protein carriers for lipids in the cytoplasm (most often across short cytoplasmic gaps between membranes that are in close contact (ie. at ER-Junctions)
What are the three classes of lipids?
- 1) Glycerolipids - has a glycerol backbone, and a hydrophilic choline
- 2) Sphingolipids - has a sphingosine instead of a glycerol backbone; some still have a choline attached like glycerolipids, while others have a sugar attached
- 3) Sterols eg. cholesterol
Explain the four steps of non-vesicular lipid transport.
- 1. LTP binds donor membrane to load cholesterol into the LTP
- 2. LTP dissociates from the donor membrane and diffuses to acceptor membrane
- 3. LTP binds the acceptor membrane, into which it unloads the cholesterol. Targetting to membranes depends on other lipids and proteins, enabling high specificity of transport
- 4. LTP dissociates from the acceptor membrane so that it can used to transport another lipid
- This is an ATP-independent process because lipids are transported down a concentration gradient.
Describe the structure of lipid transfer proteins (LTPs).
- soluble proteins - no transmembrane domains
- hydrophobic on the inside and hydrophilic on the outside
- bind lipids and proteins in target membranes
- extract lipids from membranes by burying them in a deep hydrophobic pocket
- multiple large families of LTPs - allows for trafficking of different lipids and to different intracellular locations
What is an ERJ?
- ER Junction - regions in the cell where the ER comes very close to another organelle membrane
- stabilized by bridging complexes
- distance between membranes is 10-50 nm
- membranes do not fuse
- have been identified at the plasma membrane, mitchondria, Golgi, and chloroplast
- LTP binds to both the donor and acceptor membranes simultaneously, and lipids are transported by the LTP swinging back and forth between membranes (no diffusion)
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