Phoprotein with sites of phosphorylation on residues S8, S11, S201, S204. In this study, we have made use of mutations at these sites to examine the biological consequences of Sec4p phosphorylation. Replacement of the identified phosphorylation sites with glutamic or aspartic acid residues eliminates Sec4p functionality, while introduction of alanine residues at these positions does not impact function. In this context, we interpret glutamic and aspartic acid residues to be acting as phosphomimetics. This interpretation is reinforced by the finding that substitutions of similar sized but neutral residues have no effect in these positions, and also by the fact that mutations at a selective subset of these sites gave rise to conditional sec4 mutants that accumulate vesicles at the restrictive temperature. These data suggest that phosphorylation is not necessary for Sec4p function but rather suggest that it serves to sensitize Sec4p function. To understand the mechanistic consequences of Sec4p phosphorylation we examined the ability of the protein with phosphomimetic substitutions in the positions of phosphorylated serines to undergo nucleotide binding and hydrolysis. We did not observe any significant impact of these mutations on the GTPase cycle, either intrinsically or under the influence of its known direct regulators Dss4p, Sec2p and Gyp1p. This conclusion is supported by structural considerations: the peptide regions containing the phosphorylated residues are on the opposite side of the protein to the nucleotide binding cleft and are not part of the molecule that engages the activators Sec2p and Dss4p. In contrast, our data suggest that one mechanistic consequence of in vivo Sec4p phosphorylation is to block interaction with the exocyst component effector MicroRNAs likely influence these processes by negative regulation through binding to messenger RNA targets Sec15p as phosphomimetic substitutions in these positions rendered the protein unable to interact with Sec15p but still retained interaction with another regulator, RabGDI. Sec15p action is essential for polarized exocytosis and is the only known effector of Sec4p that is necessary for viability, suggesting that the inability of the phosphomimetic Sec4p to provide function is a direct consequence of its lack of interaction with Sec15p. The exact mode of interaction between Sec4p, its effector Sec15p and other Sec15p interacting components, including the polarity establishment protein Bem1p, the Sec4p exchange factor Sec2p, the unconventional myosin Myo2p, the other seven subunits of the exocyst and the other Ras-related small GTPases that bind to the exocyst, remain to be understood. The crystal structure of the exocytic ortholog of Sec4p, Rab3A, in complex with its effector Rabphillin, demonstrates a binding mode made up of noncontiguous regions of Rab3A that includes the extended NH2terminus. In a similar fashion, the extended regions at either or both of the termini of Sec4p may constitute a binding determinant for Sec15p that can be blocked in a regulated manner by phosphorylation. Replacement of the phosphorylated residues with the negatively charged phosphomimetics may lock Sec4p in an inactive state because it can no longer form productive interactions with its effector. When not engaged in effector or other protein-protein interactions, these particular residues will be physically accessible to the action of phosphoregulatory enzymes, being located in unstructured regions that protrude from the core GTPase domain. In support of phosphorylation as a negative regulatory event on Sec4p-mediated trafficking.