Part I: Multiple choices (#1-#10)
1. Unlike soluble, cytosolic proteins, membrane proteins are more difficult to purify.
Which of the following substances is most commonly used to help purify an integral membrane protein?
(a) high salt solution
(b) sucrose
(c) detergent
(d) low pH solution
2. There are two properties of phospholipids that affect how tightly they pack together: the length of the hydrocarbon chain and the number of double bonds. The degree of packing, in turn, influences the relative mobility of these molecules in the membrane. Which of the following would yield the most highly mobile phospholipid
(listed as number of carbons and number of double bonds, respectively)?
(a) 24 carbons with 1 double bond
(b) 15 carbons with 2 double bonds
(c) 20 carbons with 2 double bonds
(d) 16 carbons with no double bonds
3. New membrane phospholipids are synthesized by enzymes bound to the _____________ side of the _________________ membrane.
(a) cytosolic, mitochondrial
(b) luminal, Golgi
(c) cytosolic, endoplasmic reticulum
(d) extracellular, plasma
4. Membrane synthesis in the cell requires the regulation of growth for both halves of the bilayer and the selective retention of certain types of lipids on one side or the other. Which group of enzymes accomplishes both of these tasks?
(a) flippases
(b) phospholipases
(c) convertases
(d) glycosylases
5. Which of the following organelles are not part of the endomembrane system?
(a) Golgi apparatus
(b) the nucleus
(c) lysosomes
(d) mitochondria
6. In which cellular location would you expect to find ribosomes translating mRNAs that encode ribosomal proteins?
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(a) in the cytosol
(b) on the rough ER
(c) the nucleus
(d) in the lumen of the ER
7. Most proteins destined to enter the endoplasmic reticulum
(a) are transported across the membrane after their synthesis is complete.
(b) are synthesized on free ribosomes in the cytosol.
(c) begin to cross the membrane while still being synthesized.
(d) remain within the endoplasmic reticulum.
8. Your friend works in a biotechnology company and has discovered a drug that blocks the ability of Ran to exchange GDP for GTP. What is the most likely effect of this drug on nuclear transport?
(a) Nuclear transport receptors would be unable to bind cargo.
(b) Nuclear transport receptors would be unable to enter the nucleus.
(c) Nuclear transport receptors would interact irreversibly with the nuclear pore fibrils.
(d) Nuclear transport receptors would be unable to release their cargo in the nucleus.
9. Which of the following phospholipid carries a net negative charge on its polar head group?
(a) phosphatidylcholine
(b) phosphatidylserine
(c) phosphatidylethanolamine
(d) sphingomyelin
10. After isolating the rough endoplasmic reticulum from the rest of the cytoplasm, you purify the RNAs attached to it. Which of the following proteins do you expect the
RNA from the rough endoplasmic reticulum to encode?
(a) soluble secreted proteins
(b) ER membrane proteins
(c) plasma membrane proteins
(d) all of the above
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Part II: Essay questions
11. Clathrin-coated vesicles bud from eukaryotic plasma membrane fragments when adaptor proteins, clathrin, and dynamin-GTP are added. What would you expect to observe if the following modifications were made to the experiment? Explain your answers.
A.
Clathrin was omitted.
B.
Dynamin was omitted.
12. Describe the role of Ran (a small GTPase) in nucleocytoplasmic transport of proteins, including both protein import and protein export.
13. Two graduate students, Mary and Jenny, are having an argument. Mary has purified a novel protein she is calling ECM1. Mary hypothesizes that ECM1 is an extracellular matrix protein because it seems to be soluble and is glycosylated. Jenny has tested the protein and found it to be N-glycosylated with a mannose 6-phosphate added to the N-linked oligosaccharide. Jenny claims that her findings should convince Mary that ECM1 is unlikely to be an extracellular matrix protein. Whom do you agree with and why?
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14.
Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to residents of the nucleus such as chromosomes, or were actively transported and accumulated regardless of their affinity for nuclear components.
A classic experiment that addressed this problem used several forms of radioactive nucleoplasmin, which is a large pentameric protein involved in chromatin assembly. In this experiment, either the intact protein or the nucleoplasmin heads, tails, or heads with a single tail were injected into the cytoplasm of a frog oocyte or into the nucleus ( Figure #14 ). All forms of nucleoplasmin, except heads, accumulated in the nucleus when injected into the cytoplasm, and all forms were retained in the nucleus when injected there.
A.
What portion of the nucleoplasmin molecule is responsible for localization in the nucleus?
B.
How do these experiments distinguish between active transport, in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion, in which a binding site for a nuclear component allows accumulation in the nucleus?
Figure #19. Cellular location of injected nucleoplasmin and nucleoplasmin components.
Schematic diagrams of autoradiographs show the cytoplasm and nucleus with the location of nucleoplasmin indicated by the shaded areas.
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15.
The Sec4 gene of budding yeast encodes a small GTPase that plays an essential role in the secretion pathway that forms the daughter bud. Normally, about 80% of the Sec4 protein is found on the cytosolic surface of transport vesicles and 20% is free in the cytosol. When temperature-sensitive Sec4 mutants of yeast ( Sec4 ts
) are incubated at high temperature, growth ceases and small vesicles accumulate in the daughter bud.
To define the role of Sec4 in secretion, you engineer two specific Sec4 mutants based on the way other small GTPases work. One mutant, Sec4 -cc
 lacks two cysteines at its C-terminus, which you expect will prevent attachment of the fatty acid required for membrane binding. The second mutant, Sec4N133I , encodes an isoleucine in place of the normal asparagine at position
133; you expect that this protein will be locked into its active state, even though it should not be able to bind GTP or GDP.
You find that Sec4-cc

binds GTP but remains entirely cytosolic with none bound to vesicles. When expressed at high levels in yeast that also have a normal Sec4 gene, it does not inhibit their growth. In contrast, Sec4N133I is located almost entirely on vesicles, and when it is expressed at high levels in normal yeast, it completely inhibits growth, and the yeast are found to be packed with small vesicles.
A.
Do you think Sec4 is required for formation of vesicles, for vesicle fusion with target membranes, or for both? Based on its function, would you guess it was analogous to mammalian Sar1 or Rab proteins?
B.
Using your knowledge of the way the analogous mammalian protein works, outline how you think normal Sec4 functions in vesicle formation and fusion. Why is some Sec4 free in the cytosol of wild-type cells? How does removal of the C-terminal cysteines prevent
Sec4- cc

from carrying out its function?
C.
Why do you think expression of Sec4N133I inhibits growth of the yeast that also express normal Sec4?
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