Article 10

Elrod-Erickson, M.J. & C.A. Kaiser (1996) Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Mol. Biol. Cell 1: 1043-1058.

Article 10 looks at the issue of sorting secreted proteins from ER and Golgi resident proteins during the secretion process. ER localized proteins such as Sec63p and Kar2p are targeted to the ER during their synthesis and maturation and are retained in the ER. At the same time, secreted proteins such as invertase that also localize to the ER remain there only transiently before being segregated to vesicles and moved progressively through the secretory pathway. Evidence outlined in the Introduction to this article suggests that secreted proteins are actively concentrated into vesicles while ER proteins are selectively removed. A short ER retention signal sequence, HDEL, has been identified. The proteins involved in cargo selectivity and fidelity are unknown.

Article 5 reported that Secl2p, Secl3p, Secl6p, and Sec23p are involved in an early step in ER to Golgi vesicle formation. Sec24p, Sec31p, and Sarlp have recently been added to this list. All are components of the vesicle coat referred to as COPII and it is thought that assembly of this complex drives vesicle formation. Both Secl3p and Sec23p have mammalian homologues suggesting that they play a central evolutionarily conserved role. Mutation of SEC13 blocks vesicle formation. The hypothesis of this study is that the secl3 block is not structural; that is, not because COPII with mutant Secl3p is structurally defective or does not assemble properly. The authors suggest the possibility that vesicle formation is blocked because some other protein(s) senses that cargo selectivity is defective and is blocking the formation/release of vesicles containing improper cargo. Such proteins would be negative regulators of vesicle formation and loss of this function should by-pass secl3 mutants.

1. Why do the authors choose seel3A rather than a secl3-ts for this suppressor selection scheme?

2. SEC13 is an essential gene. Thus, the authors could not simply construct a secl3A strain and select for suppressors. Describe the strain used for this selection and the method designed to identify secl3A by-pass suppressors. {Note: 5FOA is 5-fluoro orotic acid. In strains carrying the wild-type URA3 gene, the Ura3p enzyme converts 5FOA to a toxic compound and thus URA3 strains die on a medium containing 5FOA. In strains in which the URA3 gene is carried on a plasmid, cells that have lost the plasmid, and thus the URA3 allele, will survive. Therefore, researchers use growth on 5FOA for selecting ura3 strains.)

3. How were the potential by-pass suppressor strains screened to determine the following?

(a) That the plasmid-borne SEC13 gene was not needed for growth; that is, the suppressor mutation by-passed SEC13.

(b) That the sec!3A mutation was still present.

(c) That the suppressor mutations were recessive.

(d) That the by-pass suppressor mutations identified fell into three complementation groups. Give the genotype (for BST and SEC13 genes) and phenotype (viable/nonviable) of the parents and the resulting diploid.

4. Strains carrying the bst mutants alone were constructed by crossing the bst sec!3A mutants to wild-type (BST SEC13).

(a) Give the genotype and phenotype (leu+/leu- and viable/nonviable) of the spores in a PD, NPD, and TT tetrad. (Since the phenotype of a bst SEC13 strain would not be known at the outset of this experiment, use a ?•)

(b) If a SEC13 bst strain is viable, what would you expect to find in a PD, NPD, and TT tetrad regarding the number of viable spores and their leucine phenotype?

(c) If a SEC13 bst strain is not viable, what would you expect to find in a PD, NPD, and TT tetrad regarding the number of viable spores and their leucine phenotype?

(d) Given that a SEC13 bst strain is viable, and the viable spores in a TT tetrad were crossed to a bst secl3A strain, what results would you expect with each of the different genotypes?

(e) Given that a SEC13 bst strain is viable, and the viable spores in a TT tetrad were each transformed with an integrating plasmid that knocks out the SEC13 gene, what results would you expect with each of the different genotypes?

(f) Were both tests necessary or would just (d) or just (e) have been sufficient? Explain.

5. What novel phenotype is exhibited by the bst and bst sec!3A strains?

(a) How was this assayed?

(b) How was this novel phenotype used to clone BST11

(c) Describe the integrative disruption of BST1.

(d) What is the significance of the finding that 'the alleles of bstl isolated as suppressors of sec!3A have the same phenotype as the disrupted allele'?

6. How did the authors use their analysis of Bstlp glycosylation patterns to predict the topology of this integral membrane protein?

7. Residues of HDEL are a signal recognized by the Erd2p-dependent retention system. What result suggests that the Bst proteins are involved in a distinct retention system that is independent of the HDEL-dependent retrieval pathway?

8. Double bst mutant strains were constructed and the Kar2p secretion phenotype of the double strains was compared with the single bst mutant strains. Using the epistasis models described in Chapter 3 for a substrate-dependent pathway, suggest a functional relationship for Bstlp, Bst2p, and Bst3p.

9. The authors 'tested the ability of the bst mutations to suppress null alleles of other SEC genes in crosses segregating both a bst mutation and a null SEC allele'. Given the fact that all of the SEC genes are essential, suggest a straightforward way of demonstrating this.

10. Discuss the preferred model for Bst protein function in ER protein sorting.

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