Article 15

Gimeno, R.E., P. Espenshade, & C.A. Kaiser (1996) COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Secl6p. Mol. Biol. Cell 1: 1815-1823.

In Article 14 two-hybrid analysis and copurification studies were used to demonstrate that Secl6p binds Sec23p. In an unpublished two-hybrid study, Gimeno and Kaiser identified a Sec24p homologue that bound to the central domain of Secl6p. Sec24p is required for vesicle formation and copurifies with Sec23p as part of a large protein complex (Hicke et al., 1992). In this article, the authors explore the physical interactions of Secl6p, Sec23p, and Sec24p using two-hybrid analysis.

1. Diagram the lexA-SEC24(34-926) fusion gene construct in plasmid pRH286. Plasmid pEF202 uses the constitutive ADH1 promoter to express the bait fusion gene. Diagram the protein product encoded by this fusion gene.

2. Diagram the GAD-SEC16(565-1235) fusion gene construct in plasmid pPE167. Plasmid pGAD-GH uses the constitutive ADH1 promoter to express the Gal4p C-terminal transcription activation domain. The BamHl cloning site is located at the 3'-end of the GAD sequence. Diagram the protein product encoded by this fusion gene.

3. Diagram the LacZ reporter gene carried on plasmid pSH 18-34.

4. All three of these plasmid constructs must be introduced into the same host strain to test for the interaction of Sec24(34-926)p and Secl6(666-926)p. To do so, what nutritional gene mutations must be carried by the host strain?

5. Diagram the fusion genes bound at the promoter of the LacZ reporter.

6. The following questions are based on the results shown in Figure 1.

(a) What results indicate that the C-terminal residues of Sec24p bind to the central domain of Secl6p but not the N-terminal or C-terminal regions of Secl6p?

(b) What results indicate that the N-terminal but not the C-terminal domain of Sec24p binds Sec23p?

(c) What region of Secl6p binds Sec23p? What is the evidence?

7. How would you use two-hybrid analysis to explore the possibility that the sec 16 ts alleles described in Article 14 affect binding to Sec24p?

REFERENCE

Hicke, L., T. Yoshihisa, & R. Schekman (1992) Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum. Mol. Biol. Cell 3: 667-676.

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