Article 9

Kurihara, T. & P. Silver (1993) Suppression of a sec63 mutation identifies a novel component of the yeast endoplasmic reticulum translocation apparatus. Mol. Biol. Cell 4: 919-930.

The Sec63 protein is part of an ER-localized complex required for the translocation of secreted proteins into the ER. Additional components of this complex were identified along with SEC63 from genetic selections for mutants defective for translocation. Article 9 uses suppressor analysis to identify new components that interact physically with Sec63p. To be specific, they interact with the cytoplasmic domain of Sec63p.

1. What is unique about sec63-l compared with the other sec63 alleles (sec63-101, sec63-106, sec63-107, sec63-108)1

2. The authors give two reasons for using sec63-101 in this selection.

(a) Why is it important to have a 'tight' ts-lethal phenotype? By 'tight' the authors mean that there is no leak-through growth at the nonpermissive temperature.

(b) What is the significance to knowing that sec63-101 alters a single residue in the cytoplasmic domain?

3. Describe the library used to isolate multicopy suppressors of sec63-101. Include the type of library (genomic or cDNA), the vector, the estimated copy number of these plasmids in yeast, the site of insertion of the yeast DNA fragments, and the selection yeast marker.

4. The size of the yeast genome is about 12.6 Mbp. The average insert size of the yeast inserts in the Nasmyth library is 10 kbp.

(a) What is the minimum number of library plasmids needed to represent the entire genome?

(b) Statistically, why do you really need more than this minimum?

(c) The authors screened 6000 transformants, or by their estimate about three genomes worth. Two plasmids were isolated containing SEC63 in the insert fragment. Based on this information, is their estimate reasonable?

5. Plasmid pHSS20 contains an approximate 8 kbp yeast DNA insert which, on average, could contain three or more genes. Describe how the suppressing gene, called HSS1, was localized within this DNA fragment.

6. Figure IB demonstrates that multicopy HSS1 specifically suppresses sec63-101. For each mutant host strain, two transformants carrying either vector, pHSSl, or 'wild-type' were tested for growth at the nonpermissive temperature.

(a) What is the purpose of the 'vector' transformants?

(b) What is meant by 'wild-type' plasmid and why was this included?

(c) What result demonstrates that HSS1 only suppresses sec63-101?

7. Strains carrying a null mutation of HSS1 are temperature sensitive for growth. How was this demonstrated?

8. Describe the phenotype of the hssl A mutant versus the H SSI strain with regard to invertase and Kar2p precursor accumulation. How does this compare with sec63-101 mutant strains?

9. Does the hssl A mutant affect nuclear localization? How does this compare to sec63-101 mutant strains?

10. Define epitope tagging. Prior to this method, what methods were used to obtain antibody to a protein of interest for the purpose of subcellular localization studies or Western analysis? Discuss some of the technical drawbacks to each method.

11. Describe how the authors used in vitro mutagenesis to demonstrate the topology of Hssl protein in the ER membrane.

12. What is the mechanism of suppression (allele-specific, by-pass, or by epistasis) by which high-copy HSS1 suppresses sec63-1011 Draw a model showing Sec63p and Hsslp (Sec66p) that demonstrates the basis of this suppression.

13. The diagram below (adapted from Nelson et al., 1993) indicates the location of each alteration in Sec63 protein in the sec63 mutations. The topology of Sec63p is shown. Mutations sec63-101, sec63-102, sec63-104, and sec63-105 all have the same change, Gly511Arg. Allele sec63-108 contains two alterations, Ile431Asn and Pro503Ala. The alteration in mutation sec63-l is Alal79Thr and in sec63-107 is Pro426Asn.

sec63-108

sec63-108

Based on this diagram, are you surprised that sec63-l is not also suppressed by HSSI in high copy? Explain.

REFERENCE

Nelson, M.K., T. Kurihara, & P.A. Silver (1993) Extragenic suppressors of mutations in the cytoplasmic C terminus of SEC63 define five genes in Saccharomyces cerevisiae. Genetics 134: 159-173.

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