Article 3

Deshaies, R.J. & R. Schekman (1987) Yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J. Cell Biol. 105: 633-645.

The authors of this article are interested in identifying the genes involved in the first step in the secretory pathway, i.e. insertion into the endoplasmic reticulum (ER). In vitro studies of protein translocation in the ER using mammalian cells had been quite extensive and productive and several components of the translocation machinery had been identified (for references see Article 3). Moreover, an in vitro yeast ER protein translocation assay had recently become available. For a complete analysis of this process and determination of the mechanism of translocation, these authors undertook a genetic approach that would complement the biochemical tools.

The class A sec mutations identified in Articles 1 and 2 all appeared to affect late stages in secretion. Only mutations in SEC53 and SEC59, also isolated by this screen, affected earlier steps altering the addition of the oligosaccharide core but not translocation into the ER. In Article 3 the authors develop a new selection method specifically designed to isolate translocation-defective mutants. Their method evolved from a similar protocol used to identify such mutants in E. coli that looked for mutants that retained a normally secreted protein in the cytoplasm.

Before reading this article, it would be helpful to learn some basics regarding the genes used in the selection. Some details are reviewed in the text of Article 3 and here, but more information is available in the references listed below. HIS4 encodes a large multifunctional polypeptide that catalyzes the third (domain A of His4p), second (domain B of His4p), and tenth (domain C of His4p) steps in the synthesis of histidine. Domain 4C is histidinol dehydrogenase. This enzyme is normally cytoplasmic.

SUC2 encodes invertase, a glycosylated secreted enzyme that hydrolyzes sucrose to glucose and fructose. SUC2 produces two different sized mRNAs depending on the growth condition because different transcription start sites are used. In low glucose (<0.5%) the mRNA is longer at the 5' end and encodes an invertase with an N-terminal signal sequence. In high glucose (l%-5%) the mRNA is shorter and the encoded invertase lacks the signal sequence and remains cytoplasmic. The cytoplasmic species of invertase is synthesized in both growth conditions while the secreted form is present only in low glucose, so-called derepressed, conditions. Glycosylation of secreted invertase occurs in several steps carried out in the ER and Golgi. Thus, monitoring the extent and type of oligosaccharide additions to invertase allows one to follow its transit through the secretory process.

MFal encodes the secreted mating pheromone a-factor. The nacent product of MFal is called prepro-a-factor and consists of an N-terminal signal sequence for secretion, an approximately 60-residue region containing three potential glycosylation sites, and four tandem copies of the 13-residue a-factor. Between these four copies are spacer residues that are the sites of proteolytic cleavage used to separate the individual pheromone molecules.

Cells that are blocked in their ability to translocate protein into the ER (tunica-mycin or sec mutants) accumulate this primary translation product. Core glycosylated forms of prepro-a-factor are formed in the ER and the signal sequence is still present. Proteolytic processing begins in the Golgi but mature a-factor is seen in late secretory vesicles.

1. Figures 2A and 3A show the three protein products expressed by the two constructions SUC2 HIS4 and MFal-SUC2 HIS4. The results shown in

Figures 2B and 3B test the premise of the selection strategy.

(a) What allele of HIS4 and HOL1 is present in the host strain DYFC2-12B and why?

(b) Which of the two fusion proteins shown in Figure 2A (the one with or the one without the N-terminal signal sequence) is synthesized in DYFC2-12B transformed with SUC2-HIS4 and why? To which subcellular compartment is it localized and why? Why is this test essential in their evaluation of the potential of the selection method?

(c) The result in Figure 3B indicates that the prepro-a-factor-invertase-His4 fusion protein is unable to complement his4A. The authors propose that this is because the fusion protein is localized to the ER. How do the results in Figure 4 support this conclusion?

2. Describe the growth conditions used to select for mutants defective in ER

translocation at 30°C.

(b) In round I, 440 histidinol prototrophs were obtained and these were screened for temperature-sensitive growth on a rich medium at 37°C. Why?

(c) Why was plasmid paSHF8 cured from the five isolates which were then only to be retransformed with unmutagenized paSHF8?

3. Genetic analysis of these five isolates was complicated by the fact that, unknowingly, all were diploid. Haploid MATa and MATa strains carrying each mutant were isolated by genetic methods.

(a) Diagram the cross between a mutant MATa strain and a wild-type MATa strain. Give the genotype and phenotypes of the parents and the diploid if the mutant allele is recessive. Or dominant.

(b) Give the results of tetrad analysis of the diploid (genotype and phenotype of the four meiotic products). Assume that each mutant strain contains only a single recessive alteration and all phenotypes segregate with the single mutant allele.

(c) How might the results of the tetrad analysis differ if the mutant strain carried two different unlinked alterations if only the double mutant exhibited the mutant phenotype? If either mutant alone exhibited the mutant phenotype?

4. All the isolates carried single recessive mutations.

(a) Diagram a cross between mutants 2 and 5 showing that both are in the same complementation group. Give the genotype and phenotype of parents and diploid.

(b) Diagram a cross between mutants 2 and 5 showing that they are in different complementation groups. Give the genotype and phenotype of the parents and the resulting diploid.

(c) For the cross in part (a), show the results of tetrad analysis of the diploid.

5. The five mutants isolated in round I did not complement. Thus, they are mutations in the same gene that the authors called sec61. Round II produced an additional seven isolates all of which complemented the sec61 mutants. The authors conclude that a second gene, SEC62, has been identified. Could there be more genes with a similar mutant phenotype and how would you determine this? What is unusual about these results? Are these necessarily independent mutations?

6. Describe the phenotypes of sec61 with regard to the following proteins: cefaclor, invertase, carboxypeptidase Y. Where is CPY localized in yeast cells?

REFERENCES

Carlson, M. & D. Botstein (1982) Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell 28: 145-154.

Donahue, T.F., P.J Farabaugh, & G.R. Fink (1982) The nucleotide sequence of the HIS4 region of yeast. Gene 82: 47-59.

Julius, D., R. Schekman, & J. Thorner (1984) Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell 36: 309-318.

Keesey, J.K. Jr, R. Bigelis, & G.R. Fink (1979) The product of the HIS4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide. J. Biol. Chem. 254: 7427-7433.

Kurjan, J. & I. Herskowitz (1982) Structure of a yeast pheromone gene (MFa): a putative a-factor precursor contains four tandem copies of mature a-factor. Cell 30: 933-943.

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