706_S2006_PS6key - 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? Yes, this is possible. If there is no functional SRP, then the whole invertase protein would be made in the cytosol, and thus it would be incapable of ever getting translocated into the ER. (ii) Could your class A mutants contain a mutation in a gene encoding the SRP receptor? Why or why not? No, this is not possible. If you are detecting invertase using an antibody against its C- terminus, then an SRP receptor mutant would give no staining at all. This is because only the first ~40 amino acids of invertase would be synthesized, and SRP would then bind to its signal sequence and halt translation. This halt to translation would never be relieved, because SRP could never bind to the SRP receptor. (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. These mutants probably have mutations in COPII vesicle formation, and thus could be mutant in Sar1, COPII coat proteins like Sec23 and Sec24, or the GEF for Sar1. COPII vesicles are used for ER-to-Golgi anterograde trafficking. If the mutants fail to form COP II vesicles or fail to incorporate cargo proteins into the COP II vesicles, then you will get proteins accumulated in the ER. The mutation could also be in a gene involved in COPI vesicle formation, such as COPI coat proteins or ARF. COP I vesicles are used for retrograde trafficking of proteins from the cis-Golgi to the ER. As a result, resident ER proteins are retrieved from cis Golgi, including key proteins that are involved in COP II vesicle trafficking, such as cargo receptors, which need to be recycled after use. Defects in COP I vesicle formation will
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2 gradually deplete these key ER proteins and COP II vesicles will fail to form gradually. When new cargo proteins are synthesized, they will eventually accumulate in the ER, and
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706_S2006_PS6key - 7.06 Problem Set #6, 2006 1. The lab you...

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