Cell and Molecular biology Problem set

Cell and Molecular Biology (BIOL 4707)
Problem Set 2
(50 POINTS TOTAL)

This assignment, covering material from week 1, is due on Thursday, May 14thth at 11:59 PM to be submitted via Canvas.
• Verify that the submission was successful
• Note: The space given per question is not based on the expected size of the answer. Be sure to answer all questions asked
• Once the deadline has passed, 10% of the total assigned score will be deducted for each day it is late

You should feel free (and indeed I encourage you) to discuss the problems in study groups with your classmates, but the final answers you submit must be your own: they must be written by you in your own words and reflect your own thinking. Please read the academic integrity policy of the course.
 
1. (4 points) Consider a transmembrane protein complex that forms a hydrophilic pore across the plasma membrane of a eukaryotic cell. The pore is made of five similar protein subunits, each of which contributes a membrane-spanning  helix to form the pore. Each  helix has hydrophilic amino acid side chains on one side of the helix and hydrophobic amino acid side chains on the opposite side. Draw a possible arrangement of these five  helices in the membrane (2 points, make the location of the hydrophilic amino acids and hydrophobic amino acids clear in your drawing). Explain your reasoning (2 points, 1-2 sentences).

 
2. (6 points) You have prepared lipid vesicles (spherical lipid bilayers) that contain Na+-K+ pumps as the sole membrane protein. Assume for the sake of simplicity that each pump transports one Na+ one way and one K+ the other way in each pumping cycle, as illustrated below. All of the Na+-K+ pumps are oriented so that the portion of the molecule that normally faces the cytosol faces the outside of the vesicle. Predict what would happen under the following conditions (1 point, each prediction). Explain your reasoning (1 point, each explanation, 1-2 sentences)

a. (2 points) The solution inside the vesicles contains both the Na+ and K+ ions; the solution outside also contains both ions, as well as ATP

b. (2 points) The solution inside and outside the vesicles contains both Na+ and K+ ions, but no ATP.

c. (2 points) The solution is as in A, but the Na+-K+ pump molecules are randomly oriented, some facing one directions, some the other

 
3. (3 points) You have just joined a laboratory that is analyzing the nuclear transport machinery in yeast. Your advisor, who is known for her extraordinarily clever ideas, has given you a project with enormous potential. In principle, it would allow a genetic selection for conditional-lethal mutants in the nuclear transport apparatus.
She gave you two plasmids. Each plasmid consists of a hybrid gene under the control of a regulatable promoter (shown to the right). The hybrid gene is a fusion between a gene whose product is normally imported into the nucleus and the gene for the restriction nuclease EcoRI. The plasmid pNL+ contains a functional nuclear localization signal (NLS); the plasmid pNL– does not have an NLS. The promoter, which is from the yeast Gal1 gene, allows transcription of the hybrid gene only when the sugar galactose is present in the growth medium.
Following her instructions, you introduce the plasmids into yeast (in the absence of galactose) and then assay the transformed yeast in medium containing glucose and in medium containing galactose. Your results are shown in the table. You don’t remember what your advisor told you to expect, but you know you will be expected to explain these results at the weekly lab meeting.
Why do yeasts with the pNL+ plasmid proliferate in the presence of glucose but die in the presence of galactose (2 points, 1-2 sentences), whereas yeasts with the pNL- plasmid proliferate in both media (1 point, 1-2 sentences)?

 
4. (10 points) Translocation of proteins across rough microsomal membranes can be judged by several experimental criteria: (1) the newly synthesized proteins are protected from added proteases, unless detergents are present to solubilize the lipid bilayer; (2) the newly synthesized proteins are glycosylated by oligosaccharide transferases, which are localized exclusively to the lumen of the ER; (3) the signal peptides are cleaved by signal peptidase, which is active only on the luminal side of the ER membrane.

Use these criteria to decide whether a protein is translocated across rough microsomal membranes. The mRNA is translated into protein in a cell-free system in the absence or presence of microsomes. Samples of newly synthesized proteins are treated in four different ways: (1) no treatment, (2) addition of a protease, (3) addition of a protease and detergent, and (4) disruption of microsomes and addition of endoglycosidase H (endo H), which removes N-linked sugars that are added in the ER. An electrophoretic analysis of these samples is shown below.

a. (7 points) Using the three criteria outlined in the problem, decide whether the experimental results in the presence of microsomes (lanes 5-8) indicate that the protein is translocated across microsomal membranes (1 point, yes/no). Explain your answer (6 points, 3-4 sentences) Your explanation should discuss the differences in protein migration in each lane.

b. (3 points) Is the protein anchored in the membrane or is it translocated all the way through the membrane (1 point, anchored or translocated all the way through)? Explain your answer (2 points, 1-2 sentences). Your answer should discuss the lanes that lead you to your conclusion

 
5. (9 points) You have isolated several mutant cell lines that are defective in their ability to add carbohydrate to exported proteins. Using an easily purified protein that carries only N-linked complex oligosaccharides, you have analyzed the sugar monomers that are added in the different mutant cells. Each mutant is unique in the kinds and numbers of different sugars contained in its N-linked oligosaccharides (See table below).

a. (4.5 points) Arrange the mutants in the order that corresponds to the steps in the pathway for processing N-linked oligosaccharides, which is shown in the figure above. (Assume that each mutant cell line is defective for a single enzyme required to construct the N-linked oligosaccharide)

b. (4.5 points) Which of these mutants are defective in processing events that occur in the ER? Which mutants are defective in processing events that occur in the Golgi?

A table is provided (below) to fill in your answers.

Cell Line ER or Golgi?

6. (6 points) v-SNAREs and t-SNAREs mediate the recognition of a vesicle at its target membrane so that a vesicle displaying a particular type of v-SNARE will only fuse with a target membrane containing a complementary type of t-SNARE. In some cases, v-SNAREs and t-SNAREs may also mediate the fusion of identical membranes. In yeast cells, right before the formation of a new cell, vesicles derived from the vacuole will come together and fuse to form a new vacuole destined for the new cell. Unlike the situation we have discussed in class, the vacuolar vesicles contain both v-SNAREs and t-SNAREs. Your friend is trying to understand the role of these SNAREs in the formation of the new vacuole and consults with you regarding the interpretation of his data.

Your friend has designed an ingenious assay for the fusion of vacuolar vesicles by using alkaline phosphatase. The protein alkaline phosphatase is made in a “pro” form that must be cleaved for the protein to be active. Your friend has designed two different strains of yeast: strain A produces the “pro” form of alkaline phosphatase (pro-Pase), whereas strain B produces the protease that can cleave pro-Pase into the active form (Pase). Neither strain has the active form of the alkaline phosphatase, but when vacuolar vesicles from the strains A and B are mixed, fusion of vesicles generates active alkaline phosphatase, whose activity can be measured and quantified (Figure below).

Your friend has taken each of these yeast strains and further engineered them so that they express only the v-SNAREs, only the t-SNAREs, both SNAREs (the normal situation), or neither SNARE. He then isolates vacuolar vesicles from all strains and tests the ability of each variant form of strain A to fuse with each variant form of strain B, by using the alkaline phosphatase assay. The data are shown in the graph in Figure Q15-44B. On this graph, the SNARE present on the vesicle of the particular yeast strain is indicated as “v” (for the presence of the v-SNARE) and “t” (for the presence of the t-SNARE).

What do his data say about the requirements for v-SNAREs and t-SNAREs in the vacuolar vesicles (4 points, 3-4 sentences)? Is it important to have a specific type of SNARE (that is, v-SNARE or t-SNARE) on each vesicle (2 points, 1-2 sentences)? Answers should reference the particular experiments that justify their explanations.
7. (12 points) Fibroblast cells from patients W, X, Y, and Z, each of whom has a different inherited defect, all contain “inclusion bodies,” which are lysosomes filled with undigested material. You wish to identify the cellular basis of these defects. The possibilities are:

1. a defect in one of the lysosomal hydrolases
2. a defect in the phosphotransferase that is required for mannose-6-phosphate tagging of the lysosomal hydrolases
3. a defect in the mannose-6-phosphate receptor, which binds mannose-6-phosphate-tagged lysosomal proteins in the trans Golgi network and delivers them to lysosomes

When you incubate some of these mutant fibroblasts in a medium in which normal cells have been grown, you find that the inclusion bodies disappear. Because of these results, you suspect that the constitutive exocytic pathway in normal cells is secreting lysosomal hydrolases that are being taken up by the mutant cells. (It is known that some mannose-6-phosphate receptor molecules are found in the plasma membrane and can take up and deliver lysosomal proteins via the endocytic pathway.) You incubate cells from each patient with medium from normal cells and medium from each of the other mutant cell cultures, and get the results summarized below.

Indicate which defect (1, 2, 3) each patient (W, X, Y, Z) is most likely to have (2 points). Explain your reasoning (10 points, 4-5 sentences).