Article 4

Novick, P., S. Ferro, & R. Schekman (1981) Order of events in the yeast secretory pathway. Cell 25: 461-469.

The secretory pathway can be viewed as a substrate-dependent pathway. Genetic analysis to date has not identified negative regulators. The secreted protein is the most upstream substrate. This is then acted on by a series of Sec proteins that act on the target protein in a stepwise fashion to translocate into the ER, move it to the Golgi where it traverses the various Golgi compartments, and then package for localization to either the cell surface or the vacuole. During this process signal sequences may be removed, the target protein may be glycosylated to varying extents, and other modifications may occur. Nonetheless, because each step must be successfully completed before the next step can occur, this is a substrate-dependent pathway. In Articles 4 and 5 the authors use epistasis analysis of this substrate-dependent pathway to determine if the SEC genes lie in a single pathway (called an epistasis group) and to order the SEC genes in this pathway.

1. To carry out an epistasis analysis one must be able to distinguish differences among the phenotypes of the different mutant genes. The authors used two sec mutant phenotypes in this study.

(a) Describe the different intermediate secretory organelles accumulated in the different sec mutants.

(b) Describe the intermediates in invertase glycosylation. Include the technique used to distinguish these intermediates.

2. The SEC genes are unlinked. The cross shown below was used to construct the secl-1 sec7-l double mutant strain.

(a) Give the SEC genotype and phenotype (temperature sensitivity) of the PD, NPD, and TT tetrads derived from this diploid.

(b) Which of these haploid segregants would you choose in order to be certain that it is a double mutant?

Parents: MA7a secl-1 SEC7 his3 x MATa SEC1 sec7-l leu2 sec~ phenotype sec~ phenotype

Diploid: MATa/MATa secl-11SEC1 sec7-l!SEC7 sec+ phenotype

3. Based on the results reported here, the authors conclude that the SEC genes are in a single pathway and not separate parallel pathways.

(a) Discuss, referring to the four models of epistasis described in Chapter 6.

(b) If two of the sec mutants were in parallel pathways, a unique phenotype would have been exhibited. What do the authors suggest as this unique phenotype?

4. Which sec mutant is epistatic in each of the following gene pairs: (a) sec2-56 and sec7-11

(d) Why did the authors place SEC7 and SEC14 at the same Golgi to late secretory vesicle step and SEC2 and SEC9 at the post late secretory vesicle step?

5. Which sec mutant is epistatic in each of the following gene pairs:

(c) Why do you think the authors did not include SEC 19 in Figure 6?

(d) Where might you place it in the pathway and why?

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