We examined the known Sec4p activators, Dss4p and Sec2p on their ability to regulate recombinant phosphomimetic or phosphoablated versions of Sec4p. In this series of experiments, exchange assays were performed to examine the ability of Dss4p or Sec2p to influence the rate of GDP/GTP exchange on Sec4p, however we saw no effect between the different Sec4p alleles. Similarly, the GTPase activating protein for Sec4p, Gyp1p did not discriminate between wild type, phosphomimetic or phosphoablated versions of Sec4p. As phosphomimetic substitutions did not appear to affect the ability of Sec4p to undergo a normal nucleotide cycle, we hypothesized that phosphorylation might impact the ability of Sec4p to act in concert with its effectors. Downstream of Sec4p activation is the action of the SNARE protein Sec9p via two known effectors, Sro7p and the exocyst component Sec15p. Of these known effectors, only the action of Sec15p is essential to support cell viability. We tested the interaction between phosphomimetic Sec4p and Sec15p with the two-hybrid assay previously used to demonstrate effector interactions between Sec4p and Sec15p. The results are shown in Table 1. Wild type Sec4p and Sec4pALA showed interactions with Sec15p. In contrast, Sec15p interactions were abolished with Sec4pASP. To further investigate interactions with Sec15p, we made use of the GTP hydrolysis defective allele of Sec4p, which has been shown to have an enhanced interaction with Sec15p. The Q79L point mutation of Sec4p stimulated interaction with Sec15p, as did Sec4pALAQ79L and these interactions were absent with Sec4pASPQ79L. As previously demonstrated, Sec15p interactions required the effector domain of Sec4p; a Q79L construct where the Switch I loop was replaced with equivalent residues from Ypt1p, Sec4 EF YPT1Q79L, did not interact with Sec15p. Rab- GDP-Displacement inhibitor, a universal Rab GTPase regulator, that extracts all Rab proteins from membranes, showed equivalent interactions with all Sec4p mutants tested. Sec15p interactions were abolished when the NH2-terminal serine residues were replaced by phosphomimetics. Sec15p interactions were also abolished when the core GTPase domain was trimmed of its NH2-terminal extension while this construct showed robust interaction with Rab-GDI. These data show that in addition to the nucleotidedependency and previously identified effector region, the interaction between Sec15p and Sec4p requires peptide sequences that protrude beyond the core GTPase domain. Phosphomimetic substitutions of the phosphorylated serines in these flexible extensions blocks Sec15p interaction suggesting that phosphorylation of Sec4p is deployed in a negative regulatory mode to eliminate exocyst engagement that is crucial for successful exocytosis. To understand the effects of mutations at the individual sites of the phosphorylated serines, we then examined the effect of replacing each mutated residue in the phosphomimetic substituted protein back to the wild type serine to determine if there were individual contributions that could be analyzed for each phosphorylated serine residue. These data are shown in Figure 2a. Restoration of serine at the NH2- terminus at position 8 restored viability to the complete phosphomimetic construct. Replacement with serine at position 10 or 11 also restored viability, but the cells bearing these constructs showed a thermosensitive phenotype. Reintroduction of serine residues in either of the two COOH-terminal positions did not alter the inability of the construct to provide Sec4p function.