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Shaywitz, D.A., P.J. Espenshade, R.E. Gimeno, & C.A. Kaiser (1997) COPII subunit interactions in the assembly of the vesicle coat. J. Biol. Chem. 272: 25413-25416.

In this article, the Kaiser laboratory extends its analysis of the assembly of COPII using two-hybrid analysis. Genetic analysis identified SEC12, SEC13, SEC16, and SEC23 as interacting genes involved in ER to Golgi vesicle formation. SARI was identified as a multicopy suppressor of secl2-4 (Nakano & Muramatsu, 1989). Secl2p is reported to be a guanine nucleotide exchange factor and activates GDP release from Sarlp (Barlowe & Schekman, 1993). Secl3p was found to copurify with a 105 kDa protein that was cloned by reverse genetics and shown to be SEC31 (Salalma et al., 1997). Gimeno et al. (1996) used two-hybrid analysis to model the interaction of Secl6p, Sec23p, and Sec24p. This article adds Secl3p and Sec31p to the complex.

1. Sequences from each of the genes listed in the table below were fused to lex A using vectors pBTM116 or pGilda and used for the experiments shown in Figure 1. For each indicate the residues contained in the fusions.

Gene Residues in LexA fusion

SEC 13_

SEC24_

SEC 16_

SEC23

2. Which residues of Sec31p interact with Secl3p? What type of protein-protein interaction domain is located in this region of Sec31p?

3. Which residues of Sec31p interact with Sec23p? With Sec24p? What data support your conclusion?

4. Which residues of Sec31p interact with Secl6p?

5. Redraw Figure 4 showing only Secl3p, Secl6p, Sec23p, Sec31p, and Sec24p. Indicate which is the N-terminal and C-terminal end of Sec31p. Include in the diagram the approximate residues of Sec31p involved in the interaction with each protein.

6. Based on the table above, what information do we have on the region(s) of proteins Secl3p, Secl6p, Sec23p, and Sec24p that interact with Sec31p?

7. Secl3p and Sec31p can be purified from cell extracts as a cytoplasmic factor. This is also true of Sec23p and Sec24p and of Sarlp. These three factors can be added to ER in vitro and stimulate vesicle production. Secl6p is never found free in the cytoplasm and is very tightly bound to ER. Propose a model for the assembly of COPII. How might you test whether the Sec23p-Sec24p complex was the first to bind to Secl6p?

REFERENCES

Barlowe, C. & R. Schekman (1993) SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature 365: 347-349.

Nakano, A. & M. Muramatsu (1989) A novel GTP-binding protein, Sarlp, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J. Cell Biol. 109: 26772691.

Salama, N.R., J.S. Chuang, & R.W. Schekman (1997) SEC31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum. Mol. Biol. Cell 8: 205-217.

Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

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