Feldman, D., J. Rothblatt, & R. Schekman (1992) Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol. Cell. Biol. 12: 3288-3296.
Sequence analysis of Sec63 protein suggested that this is an integral membrane protein and, given its function in the early stages of ER translocation, Sec63p is probably localized to the ER. This must be experimentally determined. In addition the functional importance of the DnaJ-like domain needs to be demonstrated as well as its subcellular location. That is, is the DnaJ domain exposed to the cytoplasmic or lumenal side of the ER membrane? Article 8 resolves these questions using some noteworthy molecular genetic techniques.
1. Describe briefly how the anti-Sec63p antibody was obtained. Would this method be possible without the cloned SEC63 gene? Can it, or a related method, be applied to any cloned gene?
2. Describe the position of the ER in yeast.
3. Why is it important for the authors to demonstrate that overproduction of Sec63p does not result in mislocalization?
4. Discuss the results in Figure 3.
(a) Indicate the effect of each of the different treatments on soluble, membrane-associated, and integral membrane proteins.
(b) Why was Sec62p included in this experiment?
(c) Why was Sec23p included in this experiment?
5. The topology of Sec63p was explored using sensitivity to protease digestion and constructing Sec63-Suc2 fusion proteins. Both methods lead to the same conclusion.
(a) Diagram the protein products produced by the fusion constructions A28, A529, A608, A610, and the vector construct. Use the diagram in Figure 5A as a guide.
(b) Sequence analysis in Article 7 indicated that there are three potential glycosylation sites just after (C-terminal to) the third hydrophobic region. Based on the results in Figure 5B lanes 1 and 2, is Sec63p protein glycosylated at these sites?
(c) Could this analysis have been done with invertase if it were not known to be glycosylated?
(d) How might this analysis been done with SEC63 HIS4 fusions?
6. SEC63 mutations of two types were constructed. The first consisted of a series of in-frame SEC63-SUC2 fusions to different sites in the SEC63 ORF. The second series were point mutations in SEC63 altering a single residue in the DnaJ domain. Both were tested for their ability to complement sec63 mutations. The results are given in Figure 5.
(a) Describe the method used to test complementation of the sec63-l temperature-sensitive allele.
(b) SEC63 is an essential gene. Thus, strains carrying the sec63A null allele are inviable. Complementation of sec63A by the mutant allele, which could be null alleles, using the method described in part (a) is not possible. Describe the method used to test complementation of the sec63A null allele.
(c) Just prior to the publication of this article, the genes encoding several DnaJ domain-containing proteins were sequenced. What role did this play in the mutation analysis described here?
7. The authors propose that the DnaJ domain of Sec63p is required for its function in protein translocation. Why is it important for their argument to demonstrate that Sec63p complex formation is normal in the DnaJ domain mutants?
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