1. The studies of Palade and colleagues used pulse-chase labeling with radioactively labeled amino acids and autoradiography to visualize the location of newly synthesized proteins in pancreatic acinar cells. These early experiments provided invaluable information on protein synthesis and intercompartmental transport. New methods have replaced these early approaches, but two basic requirements are still necessary for any assay to study this type of protein transport. What are they, and how do recent experimental approaches meet these criteria?

2. 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 the pinching off of vesicles?

3. Treatment of cells with the drug brefeldin A (BFA) has the effect of uncoating Golgi 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 ARF might have the same effect as treating cells with BFA.

4. Microinjection of an antibody known as EAGE, which reacts with the “hinge” region of the β subunit of COPI, causes accumulation of Golgi enzymes in transport vesicles and inhibits anterograde transport of newly synthesized vesicles from the ER to the plasma membrane. What effect does the antibody have on COPI activity? Explain the results.

5. 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?

6. Sec18 is a yeast gene that encodes NSF. Mutations of this gene produce class C mutants. 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?

7. What feature of procollagen synthesis provided early evidence for the Golgi cisternal maturation model?

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-maturation 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? What observation regarding AP3 suggests that clathrin is 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: the 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 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 lysosomes with the packaging of proteins into regulated secretory vesicles such as those containing insulin. Compare and contrast the sorting of proteins to the basolateral versus apical cell surfaces in MDCK cells and in hepatocytes.

12. What does the budding of influenza virus and vesicular stomatitis virus (VSV) from polarized MDCK cells reveal about the sorting of newly synthesized plasma membrane proteins to the apical or basolateral domains? Now consider the following result: a peptide with a sequence identical to that of the VSV G protein cytoplasmic domain inhibits targeting of the G protein to the basolateral surface and has no effect on HA targeting to the apical membrane, but a peptide in which the single tyrosine residue is mutated to an alanine has no effect on G protein basolateral targeting. What does this tell you about the sorting process?

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13. Describe the role of pH in regulating the interaction between mannose 6-phosphate and the M6P receptor. Why does a rise in 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 an 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 a portion of the HIV Gag protein in a synthetic peptide. Which portion of the HIV Gag protein would be a logical choice? What normal cellular process might this inhibitor block?

15. The phagocytic and autophagic pathways serve two fundamentally different roles, but both deliver their vesicles to the lysosome. What are the fundamental differences between the two pathways? Describe the three basic steps in the formation and fusion of autophagosomes.

16. Compare and contrast the location and pH sensitivity of receptor-ligand interaction in the LDL and transferrin receptor-mediated endocytosis pathways.

17. What do mutations in the cytoplasmic domain of the LDL receptor that cause familial hypercholesterolemia reveal about the receptor-mediated endocytosis pathway?