1. 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
  2. What are the functions of protein coats?
    • large protein cages that are spherical in shape
    • 1) recruit cargo
    • 2) drive vesicle formation - requires energy
  3. 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
  4. 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
  5. 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.
  6. 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
  7. 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
  8. 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
  9. 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 
  10. 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
  11. 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
  12. 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
  13. 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)
  14. 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
  15. 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.
  16. 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
  17. 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)
Card Set
ANAT390 lecture 5 vesicles lysosomes and lipids