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Roberg, K.J., S. Bickel, N. Rowley, & C.A. Kaiser (1997) Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. Genetics 147: 1569-1584.

The initial goal of this study was to identify new genes encoding proteins involved in ER to Golgi vesicle formation. Mutations in SEC13 were found to produce reduced numbers of 50 nm vesicles (ER to Golgi intermediates) and to exhibit synthetic lethal interactions with mutations in SEC12, SEC16, and SEC23 (Kaiser & Schekman, 1990). This approach might have identified nonessential components of the complex or factors involved in the assembly or stability of the complex whose function became essential when an essential component is defective.

1. Describe the genetic screen used to isolate mutations that exhibit synthetic lethality with seel3-1.

2. The 139 nonsectoring colonies were tested to identify clones carrying mutations of interest, i.e. those that contained secl3-l synthetic lethal mutations and exclude uninteresting or complex multiple changes.

(a) Why were 82 colonies excluded from further analysis based on the fact that the introduction of a plasmid carrying SEC13 did not allow sectoring?

(b) The remaining 57 nonsectoring colonies were crossed to a secl3-l strain. Most, 52, showed 2:2 segregation of sectoring indicating a single 1st mutation. Diagram the cross.

(c) The five nonsectoring colonies eliminated in (b) had multiple alterations. Assuming two alterations to produce the nonsectoring phenotype, what would be the phenotype of the spores in a TT tetrad?

3. Diagram the crosses used to place the 1st mutations into complementation groups.

4. Mutations in SEC12, SEC16, and SEC23 were expected. How did the authors screen the 1st mutant strains to determine if they carried alterations in these genes?

5. The authors wanted to determine whether Istl exhibited synthetic lethal interactions with other sec genes.

(a) Diagram the cross used to separate Istl from seel3-1.

(b) Diagram the cross and tetrad analysis used to test Istl for synthetic lethal interaction with secl6-2.

6. Based on the results shown in Table 2, the 1st mutations were placed in two classes. Discuss why LST1 and LST6 were not considered further in this study.

7. Describe briefly the method used to clone LST5. How was it determined to be THR4 encoding threonine synthetase?

8. Explain in your own words why a mutation in a gene encoding an enzyme for the biosynthesis of an amino acid would exhibit synthetic lethality in combination with seel3-1, a mutation affecting the late secretory pathway.

9. Which amino acid permeases are dependent on Secl3p for delivery from the Golgi to plasma membrane?

10. How do the results in Figure 2 distinguish seel3-4 from seel3-1, seel3-5, and seel 3-71

(a) If the authors had used secl3-4 in their screen, would they have isolated synthetic lethal mutations in the same LST genes as with sec 13-1 and if so why or why not?

(b) The authors examined this question: Does secl3-4 cause synthetic lethality with lst4-l and lst7-ll What experiment was done and what were the results?

11. Figure 3 compares the effect of secl3-l, secl2-4, secl6-2, sec23-l, and sec31-l on citrulline uptake (a measure of Gapl permease activity). Only seel3-1 reduces Gaplp activity. Why would it have been valuable to test all the available mutant alleles of each gene? This is particularly true for SEC16. Why? (Hint, based on 1st genes identified by their screen.)

12. The authors used the analogues listed in Figure 4 as a quick assay of specific permease activities. Why is resistance to the analogue an indicator of reduced transport activity?

13. How did the authors demonstrate that lst4, lst7, and lst8 mutations specifically affect the secretion of certain amino acid permeases and do not cause general defects in secretion?

14. In the experiment shown in Figure 6, why did the authors determine the position in the gradient of Sec61p, GDPase, and plasma membrane ATPase? The results in Figure 6 are consistent with those in Figure 7 with regard to the subcellular distribution of Gaplp in lst4, lst7, and lst8 mutant strains. Describe.

15. Why did the authors decide to test pepl2A as a possible suppressor of seel3-1, lst4, lst7, and 1st HI How might you use genetic analysis to identify additional mutations in genes involved in Golgi to vacuole transport?

16. The authors suggest that Lst4p, Lst7p, and Lst8p might play a direct role in Golgi to plasma membrane transport of Gaplp, that is, that these are components of a Secl3p-containing complex or are involved in the function or assembly of such a complex. An alternate hypothesis is that they are involved in nitrogen sensing. LST7 and LST8 have both been cloned. How might you use the tools of molecular genetics to explore these possibilities?

REFERENCE

Kaiser, C.A. & R. Schekman (1990) Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell 61: 723-733.

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