Sec15p interactions with Sec4p are abbrogated by phosphomimetics.

The interaction between Sec15p, Gdi1p and Sec4p was assessed using the yeast two-hybrid system. Reporter cells were cotransformed with a prey vector encoding SEC15 and a bait plasmid encoding either wild type or GTPase deficient mutant Sec4p Q79L with phosphorylated serine residues replaced with either alanine or aspartic acid. The strength of the interaction was determined using a β-galactosidase reporter assay and assigned a relative strength of (++++, strong interaction to −, no interaction).