Figure 6.

Mutations in SEC9 H1 and H2 but not in SNC2 or SSO1 act as dominant negative alleles when overexpressed, but heterodimerization with wild-type Sec9 is not a likely mechanism for dominant negativity. (A) Each of the alleles was subcloned into a GAL1 expression plasmid and introduced into a wild-type yeast strain by integrative transformation. Transformants were replica-plated on YPD or YP-Gal plates and incubated at the indicated temperatures for 2–3 d. (B) GST–Sec9 fusion proteins were tested for their ability to interact with excess-soluble Sec9. Binding reactions were performed as described in MATERIALS AND METHODS. Pellets containing bound proteins (P) were separated from unbound proteins (S) by centrifugation and subjected to the Western blotting with α-Sec9 antibody.