Real problem 3 PDF

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problem set 3...

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BICD 110 Problem Set #3

1. Use the letters (A-F) below for the best answer to each question. (i)

Tom/Tim protein complexes are involved in

(ii)

Importin proteins are involved in A

(iii)

Ran GEF protein is involved in F

(iv)

FG Nup proteins are involved in

(v)

ATP hydrolysis is involved in E

A. B. C.

B.

. . A. .

Protein translocation into the nucleus. D. Ran GTPase activation. Protein translocation into mitochondria. E. B & C are both correct. Preventing protein folding prior to organelle import. F. A & D are both correct.

2. Where will proteins that lack any targeting sequence ultimately reside?

A) Proteins without any targeting sequence reside in the cytosol. 3. The protein sorting decisions for import into mitochondria or into nuclei are both made: A. x B. C. D. E.

During cotranslation translocation at the rough ER. During nascent polypeptide translation in the cytosol. By diffusion at the import pores. After proteolytic cleavage of an import sequence. By the membrane potential at each organelle.

4. Transmembrane ion channels involved in maintaining the cell resting membrane potential are inserted into the ER membrane lipid bilayer. x A. Cotranslationally. B. Post-translationally. C. Pretranslationally. D. Electrogenically. E. with the ion gradient. 5. Targeted delivery of most secretory proteins at the ER is powered by: A. ATP hydrolysis by an ATPase pump. B. Protein disulfide isomerase. C. Gycosyltransferases. x D. GTP hydrolysis by SRP and SRP receptor, and translation. E. ATP hydrolysis by Hsc70 chaperones

6. Where is it made, and where will it go? For each polypeptide with the following

features below (a-f), name specifically where in the cell each polypeptide would be (i) actively translated and (II) where it is targeted upon completion of translation. Be as specific as possible for (lumen, mitochondrial membrane, secreted, etc). Polypeptide has: a)

b)

c) d)

(i) translated? RER

RER

(ii) localized? ER plasma membrane

(ER lumen) secreted

ribosomes in cytosol

nucleus

cytosol,ribosomes, matrix of mitochondria

7. A newly discovered intracellular bacterial pathogen produces a large protein virulence factor responsible for damaging host cell mitochondrial functions following infection. Some researchers suggest that the bacterial protein acts directly in mitochondria, but you suspect the protein acts elsewhere with an indirect effect on mitochondria. What are two predictions about the bacterial protein if it acts directly inside host cell mitochondria? And, how would you test both predictions?

A) Two predictions are that the protein would have an N-term matrix sequence and it would interact with the Tom20 or Tom22 receptor. You would test the first prediction using immunofluorescent microscopy, or differential centrifugation. To test if it is in mitochondrial matrix sequence and the second prediction you could perform a GFP tracking experiment to determine localization or side directed mutation sequences to see if it is located in the mitochondria.

8.

You suspect that a protein of interest normally shuttles between the nucleus and cytosol, even though immunofluorescence shows mostly a cytoplasmic distribution. If the protein shuttles, what are two predictions about this protein, and how can you experimentally validate them? A) Two predictions are that it would have both an NLS and an NES sequence. To validate this a mutational analysis on the NLS and NES sequence would be done. This would be done by mutating the charged lysines and glycines on NLS, For NES sequence a hydrophobic residue could be removed to see if import and export function is changed.

9.

Splice variants of a transmembrane domain protein are fused to alkaline phosphatase (AP), allowing you to measure activity of this enzyme depending if it is expressed on the surface of microsomes or not (you can’t measure the activity of the enzyme if it’s inside the microsomes). After performing this, you also obtained hydropathy plots telling you that regions A-E are hydrophobic sequences of 20-30AA long. The following results were obtained:

a) Draw the orientation of each of these proteins in the microsome, clearly labeling the cytosolic side in each case.

For polypeptides 1-3, the hydrophobic sequences would be on the inside of the membrane while the positive charges are in the cytosol, and the N-terminus is in the cytosol. For polypeptide 1, the C-term is in the lumen while for polypeptides 2 and 3, the C term is in the cytosol.

b) For each protein above (1-3), indicate where the positively charged AA would be located on the sequences above (draw them in) or describe where they would be located assuming ALL A-E are SA sequences. Remember splice variants can have different amino acid sequences in any part of the sequence. Do not assume the sequence surrounding each hydrophobic region above is the same. A) a. 1. Before A, after B, before C, after D, before E b. 2. After D, before C, after B, before A c. 3. After E, before D, after C, before B

c) Draw what the hydropathy plot looked like for protein #1

d) In order to confirm your topology predicted in A, you use site directed mutagenesis to generate three different constructs, each with a novel N-linked glycosylation site that you created shown below. Draw the expected SDS-PAGE results for each of the three constructs showing the size of the protein with and without EndoH treatment.

10. Based on the description below, list 2 different types of loss of function (LOF) mutant proteins for each description that could cause the following phenotypes in a temperature sensitive mutant yeast strain when grown at the non-permissive temperature. a. Accumulation in the cytosol of secretory proteins A) SRP/SRP receptor; Translocon (Sec61)

b. Accumulation in the cytosol of mitochondrial targeted proteins a) N-term matrix sequence; Tom & Tim; chaperone (hsc70) c. Accumulation in the nucleus of a protein that gets shuttled between the nucleoplasm and the cytosol a) NES; exportin; Ran-GDP

11. You want to better understand protein disulfide isomerase (PDI) localization and function for generating protein disulfide bonds. You create two different GFP-fusion constructs to express and monitor tagged PDI in the cell, PDI:GFP and GFP:PDI, where GFP sequence has been added to either the C-term or N-term of PDI, respectively. After you transfect different cell populations with the two DNA constructs you test for PDI transgene protein expression by anti-GFP Western Blot and then examine localization by microscopy. (Note: transfection is a way to get DNA into the cells so it can be expressed) You know that PDI encodes for a 57 kDa protein and GFP is 27 kDa. You are surprised to see that while both protein fusion constructs are expressed at the expected molecular weight and at similar levels, you get different results in localization…!? Ah! That’s it! Explain what you observe, and why you recovered similar protein expression but distinct localization for the two PDI constructs. A) Protein localization depends on if the GFP is bound (C or N terminal) of the PDI construct, which depends on the signaling sequence. PDI:GFP is free on the N-terminus thus it can enter through the ER lumen while GFP:PDI has ERS signaling on the N-terminus so it cannot enter on the ER lumen. This is blocking its transmission. This accounts for differences in localization....