706_S2006_PS6 - 7.06 Problem Set #6, 2006 1. The lab you...

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1 7.06 Problem Set #6, 2006 1. The lab you are working in has a big collection of temperature-sensitive secretion mutants (“sec” mutants), containing mutants belonging to Classes A, B, C, D and E. When you transfer the mutants from permissive to non-permissive temperature, they accumulate secreted proteins (such as invertase) at different points along the secretion pathway (as shown in Figure 17-5 in your textbook, page 706). You have categorized each of the mutants you have into the appropriate class (for instance, you categorized each class A mutant that way because a known secreted protein accumulated in the cytosol in those mutants). Keep in mind that temperature sensitive mutations normally eliminate the function of the gene at high temperatures. (a) For this part of the problem only, assume that you categorized which mutants were in each class by doing immuno-gold EM using an antibody made against just the C-terminal end of invertase. (i) Could your class A mutants contain a mutation in a gene encoding the RNA portion of SRP? Why or why not? (ii) Could your class A mutants contain a mutation in a gene encoding the SRP receptor? Why or why not? (b) Give three examples of proteins that, when mutated, will give you the Class B phenotype. Explain briefly why this kind of mutation in each protein you named would result in a Class B phenotype. (c) You take several Class C mutants and do a detailed time-course analysis on each one. Immediately after you shift the yeast mutants to non-permissive temperature, you start taking time points. You collect the recently shifted yeast, and analyze the vesicles accumulating in those yeast using immuno-gold EM analysis with an antibody against the coat protein that should initially surround the accumulating vesicles. (i) Which coat protein is that? (ii) Are these vesicles traveling in the anterograde or retrograde direction? (d) You do the experiment described in part (c) . You observe the following three sub-classes of mutants. Subclass 1: Vesicles accumulate quickly and have coats Subclass 2: Vesicles accumulate quickly but do not have coats Subclass 3: No vesicles accumulate at first, but later, you see accumulated vesicles
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2 For each subclass, give an example of a protein that, when mutated, will give you this specific subclass of the Class C phenotype. Explain briefly why this kind of mutation in each protein you named would result in this specific subclass of the Class C phenotype. 2. You are studying a novel cell-surface receptor protein that is embedded in the plasma membrane of yeast. Analysis of the amino acid sequence of the polypeptide reveals that there are three stretches of hydrophobic residues of about 25 amino acids each. Additionally, mass spectrometry analysis reveals that the C-terminus of your protein is glycosylated. (a)
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_S2006_PS6 - 7.06 Problem Set #6, 2006 1. The lab you...

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