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Scidmore, M.A., H.H. Okamura, & M.D. Rose (1993) Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum. Mol. Biol. Cell 4: 1145-1159.

The E. coli DnaJ and DnaK (an HSP70 homologue) proteins interact physically. The authors of this article ask the question: Do Sec63p (DnaJ homologue) and Kar2p (DnaK homologue) also interact physically? The DnaJ domain of Sec63p is located in the lumen of the ER and Kar2p is the only yeast DnaK homologue localized to the ER lumen. The authors undertake a genetic approach to this question. They will use allele-specific genetic interactions, suppression, and enhancement to reveal a direct physical interaction.

1. sec63-l is the only mutation available that affects a residue in the lumenal DnaJ domain of Sec63p. sec63-101 alters a residue in the C-terminal cytoplasmic domain of Sec63p. Table 2 shows the results of tetrad analysis of crosses between kar2-159 and mutant alleles of SEC61, SEC62, and SEC63.

(a) What is the purpose of crosses 1 and 2?

(b) What results indicate an allele-specific synthetic lethal interaction between kar2-159 and secó31 Include in your answer the genotype and expected phenotype (spore viability at 23°C) of a PD, NPD, and TT tetrad where synthetic lethality is exhibited.

(c) What is the significance of the result obtained at a germination temperature of 13°C?

(d) What results demonstrate the lack of enhancement between kar2 and secól or sec62? From this result can you conclude that Kar2p does not interact with either Sec61p or Sec62p and why?

(e) The authors state, '. . . none of the kar2 Ts-alleles tested were synthetically lethal with sec61-l, sec62-l, sec65-l, sec63-101 nor sec63-106...'. What additional information regarding the interaction (or potential interaction) between Kar2p and Sec61p, Sec62p, Sec65p, and Sec63p is provided by this statement?

2. Table 4 presents the results of crosses between sec63-l and several kar2 alleles to test the allele-specificity of the interaction.

(a) Examine the results of crosses 1, 2, 3, 4, 6, 8, 10, 12, and 14 in which the spores were germinated at 23°C. Do you find evidence of an allele-specific synthetic lethal interaction? Why or why not?

(b) Comparison of the results of crosses 6, 8, 10, and 12 in which the spores were germinated at 13°C does suggest allele-specificity. Discuss. Which kar2 alleles exhibit synthetic lethality with sec63-l and which do not?

(c) Complete the diagram below proposing the location of the altered residues in kar2-133, kar2-203, kar2-157, and kar2-191.

3. Describe the genetic screen designed to isolate dominant mutations in KAR2 capable of suppressing the temperature-sensitive growth phenotype of sec63-l. The authors showed that KAR2 in high copy does not suppress sec63-l. How would it have complicated the screening procedure if they had found that KAR2 overexpression did suppress sec63-P.

4. Suggest a method of determining the phenotype of these KAR2 dominant alleles in combination with wild-type SEC63. If some of these alterations affect Sec63p-Kar2p binding, what phenotypes might you expect for these KAR2 alleles? Into what two classes does the KAR2 dominant suppressor fall? Might you expect different phenotypes for these two classes of suppressors in this analysis and if so why?

5. Table 5 demonstrates that the dominant KAR2 suppressors of sec63-l are allele specific. These results taken together with the results shown in Table 4 indicating partial allele-specific synthetic lethality between sec63-l and kar2 led the authors to propose that, like other DnaJ/DnaK pairs, Sec63p and Kar2p interact physically. Moreover, based on studies of other DnaJ/DnaK pairs, they suggest that this interaction stimulates the ATPase activity of Kar2p. These conclusions would be greatly strengthened by some biochemical assays of Sec63p-Kar2p binding and Kar2p ATPase activity using the different mutant alleles of these proteins.

(a) In the Discussion the authors allude to a copurification technique developed by Brodsky and Schekman that was able to demonstrate the complex formation between Sec63p and Kar2p but not Sec63-lp and Kar2p. Using this method, you explore the physical interaction between the proteins encoded by sec63-l and the different dominant KAR2 suppressor. What result would you expect if the hypothesis of a direct interaction between Sec63p and Kar2p and the authors' explanation of the mechanism of suppression by the KAR2 dominant suppressors were correct? Use the table below for your answer. The first two rows are controls to demonstrate that you are carrying out the binding assay correctly.

sec63-l sec63-l

sec63-101

SEC63 allele

KAR2 allele

Copurification with

ATPase activity of

Sec63 protein

purified Kar2 protein

SEC63

KAR2

Low

sec63-l

KAR2

Low

sec63-l

KAR2-699

Low

sec63-l

KAR2-6199

Low

sec63-l

KAR2-6116

Low

sec63-l

KAR2-6139

Very high

(b) Would the Kar2p dominant suppressor proteins be expected to copurify with wild-type Sec63p?

(c) Develop a method to purify Kar2 protein from strains expressing wildtype and each of the four mutant alleles listed in the above table and assay ATPase activity in vitro. Your results are shown in the table above. Propose a mechanism for suppression of sec63-l by KAR2-6139.

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