1. The studies of Palade and colleagues using pulse-chase labeling with radioactively labeled amino acids and autora-diography to visualize the location of the radiolabeled proteins is a classic case of the experimentalist realizing what works well in his or her biological system. These experiments were done with pancreatic acinar cells. Alternatively, HeLa cells can be used. HeLa cells are a classic human cell line originating from a cervical carcinoma. When applied to HeLa cells, the same experimental protocols are a dismal failure with respect to tracking secretion. What would you expect autoradiography of a HeLa cell to look like after pulse-labeling with radioactive amino acids?
2. Sec18 is a yeast gene that encodes NSF. It is a class C mutant in the yeast secretory pathway. What is the mechanistic role of NSF in membrane trafficking. As indicated by its class C phenotype, why does an NSF mutation produce accumulation of vesicles at what appears to be only one stage of the secretory pathway?
3. Vesicle budding is associated with coat proteins. What is the role of coat proteins in vesicle budding? How are coat proteins recruited to membranes? What kinds of molecules are likely to be included or excluded from newly formed vesicles? What is the best-known example of a protein likely to be involved in vesicle pinching off?
4. Treatment of cells with the drug brefeldin A (BFA) has the effect of decoating Golgi apparatus membranes, resulting in a cell in which the vast majority of Golgi proteins are found in the ER. What inferences can be made from this observation regarding roles of coat proteins other than promoting vesicle formation? Predict what type of mutation in Arf1 might have the same effect as treating cells with BFA.
5. An antibody to an exposed "hinge" region of pCOPI known as EAGE blocks the function of pCOPI when mi-croinjected into HeLa cells. Predict what the consequences of this functional block might be for anterograde transport from the ER to the plasma membrane. Propose an experiment to test whether the effect of EAGE microinjection is initially on anterograde or retrograde transport.
6. Specificity in fusion between vesicles involves two discrete and sequential processes. Describe the first of the two processes and its regulation by GTPase switch proteins. What effect on the size of early endosomes might result from overexpression of a mutant form of Rab5 that is stuck in the GTP-bound state?
7. Two different protein-mediated membrane fusion processes are described in this chapter, SNARE- and viral HA-mediated fusion. Compare and contrast the two. In each example give particular attention to what the direct effect of polypeptide sequences in membrane fusion is and to what controls the specificity of membrane fusion in each.
Rab proteins 711 receptor-mediated endocytosis 728 regulated secretion 724 retrograde transport 715 sec mutants 706 secretory pathway 701 sorting signals 711 synaptotagmin 737 transcytosis 727 trans-Golgi network
(TGN) 701 transport vesicles 701 t-SNAREs 708 v-SNAREs 708
8. Sorting signals that cause retrograde transport of a protein in the secretory pathway are sometimes known as retrieval sequences. List the two known examples of retrieval sequences for soluble and membrane proteins of the ER? How does the presence of a retrieval sequence on a soluble ER protein result in its retrieval from the cis-Golgi complex? Describe how the concept of a retrieval sequence is essential to the cisternal-progression model.
9. Clathrin adapter protein (AP) complexes bind directly to the cytosolic face of membrane proteins and also interact with clathrin. What are the four known adapter protein complexes? Why may clathrin be considered to be an accessory protein to a core coat composed of adapter proteins?
10. I-cell disease is a classic example of an inherited human defect in protein targeting that affects an entire class of proteins, soluble enzymes of the lysosome. What is the molecular defect in I-cell disease? Why does it affect the targeting of an entire class of proteins? What other types of mutations might produce the same phenotype?
11. The TGN, trans-Golgi network, is the site of multiple sorting processes as proteins and lipids exit the Golgi complex. Compare and contrast the sorting of proteins to lyso-somes versus the packaging of proteins into regulated secretory granules such as those containing insulin. Compare and contrast the sorting of proteins to the basolateral versus apical cell surfaces in MDCK cells versus hepatocytes.
12. The efficiency of bacterial phagocytosis by macrophages is increased greatly by first binding antibody molecules to the bacterial surface. On the basis of prior descriptions of antibody structure, to what portion of the immunoglobulin molecule do you predict a macrophage receptor for antibody bound to bacteria might be directed? Design an experiment to test this prediction.
13. Describe how pH plays a key role in regulating the interaction between mannose 6-phosphate and the mannose 6-phosphate receptor. Why does elevating endosomal pH lead to the secretion of newly synthesized lysosomal enzymes into the extracellular medium?
14. What mechanistic features are shared by (a) the formation of multivesicular endosomes by budding into the interior of the endosome and (b) the outward budding of HIV virus at the cell surface? You wish to design a peptide inhibitor/competitor of HIV budding and decide to mimic in a synthetic peptide a portion of the HIV Gag protein. Which portion of the HIV Gag protein would be a logical choice? What normal cellular process might this inhibitor block?
15. The exocytosis of neurotransmitter-filled synaptic vesicles is an example of regulated exocytosis. How is the influx of Ca2+ following arrival of an action potential at the axon terminus sensed and linked to the exocytosis of synaptic vesicles? Why do normal Drosophila neurons incubated in the presence of a nonhydrolyzable analog of GTP have the same appearance as nerve terminals in shi mutants?
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