Espenshade, P., R.E. Gimeno, E. Holzmacher, P. Teung, & C.A. Kaiser (1995) Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p. J. Cell Biol. 131: 311-324.
SEC12, SEC13, SEC16, and SEC23 are required for vesicle formation for ER to Golgi transport. This article begins to explore the role of the encoded proteins, in particular Secl6p.
1. Review the biochemical functions of Sarlp, Secl2p, and Sec23p. What evidence suggests that S EC 16 genetically interacts with SARI?
2. Describe the strategy used to isolate SEC16. Include
(a) a description of the library;
(b) the genotype and phenotype of the host strain;
(c) the phenotype used to select transformants containing possible SEC16 plasmids.
3. Given the library used for the isolation, why is it unlikely that multicopy suppressors of secl6-l were isolated? Targeted integration was used to definitively demonstrate that the cloned 7.2 kbp BamHI-Sphl fragment contained the real SEC16 gene. Describe how this was done; include a diagram of the cross.
4. 'Each mutation was mapped by in vivo recombination tests with a nested set of SEC16 deletions' according to the method of Falco et al. (1983).
(a) Describe this method. Use diagrams where needed for clarity.
(b) How was the nested set of deletions in SEC16 constructed (see Methods and Materials)?
5. Gap repair was used to clone the secl6 mutations. Describe how you would do this for secl6-5. Include in your answer the basis for you selection of the appropriate plasmid to use for the gap repair.
6. Given the size of Secl6p, 2194 residues, what can you conclude about its function based on the location of the five secl6 mutations?
7. Secl6p is a very large protein with an essential function. The experiments presented in Figure 2 test whether all parts of this protein are essential by determining if truncated alleles are able to complement secl6 mutations.
(a) What important difference is there between the vectors in plasmids pPE8 and pPE129?
(b) Different methods were used to test the ability of the plasmids to complement seel6-1 and seel6-1 A. Describe both.
(c) Do these results demonstrate that the C-terminal 30 residues are essential or that they are essential in the absence of N-terminal residues 1-565?
(d) Propose an explanation for the finding that pPE129 complements all three secl6 mutant alleles but pPE130 only complements secl6-l and secl6-2 and not sec 16-1 A. (Hint: think about intragenic complementation.)
following questions relate to Figure 3.
Describe how Secl6p depletion was achieved. Include a description of the GALlpro-SEC16 fusion gene.
Lanes 7, 8, and 9 represent the positive control condition. Discuss. Include a description of the maturation of CPY.
Lanes 13, 14, and 15 represent the negative control condition. How do these results demonstrate that CPY does not exit the ER in secl6-2 cells at the nonpermissive temperature?
GAL1 is a glucose-repressed gene but lanes 1, 2, and 3 demonstrate that there is some expression of the GALlpro-SEC16 fusion gene in cells grown in 1% glucose and 1% galactose. How do we know this? What results demonstrate that both depletion and overproduction of Secl6p block CPY maturation at the ER? What is the significance of this finding?
(a) The constructs in Figure 2 do not provide a yeast promoter. Instead, 'transcription presumably was initiated within vector sequences'. How does the transcription expression of the SEC16 constructs in Figure 5 differ from those in Figure 2?
(b) Based on the results with plasmids pPE46 and pPE27 where would you locate the dominant negative domain?
(c) Why was the result with plasmid pPE53 unexpected? How do the authors explain this? Propose another explanation based on your finding that overproduction of a fragment containing residues 650-1050 from the GAL1 promoter does not allow growth on galactose.
10. Present a model explaining why both depletion of Secl6p and overproduction of Secl6p cause a block in secretion.
Falco, S.C., M. Rose, & D. Botstein (1983) Homologous recombination between episomal plasmids and chromosomes in yeast. Genetics 105: 843-856.
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