Characterization of Sec3 and its conformation within the exocyst complex

Abstract

For eukaryotic cells to function properly, vesicles need to be targeted to proper partner membranes. Exocyst is a heteroctameric complex that acts as a platform for a suite of regulatory proteins to guide vesicle targeting and fusion. A key interaction is with SNAREs, which provide the mechanical force to fuse membranes. Several exocyst components have been implicated in SNARE regulation, but only the Sec3 subunit has been shown to play a role in driving SNARE assembly. The N-terminal region of Sec3 (Sec3N) is highly flexible and partially unstructured which has made characterizing Sec3N challenging, especially its position relative to the rest of the exocyst complex. The extent to which the mostly unstructured nature of Sec3N plays a role in exocyst function, particularly SNARE-binding, is unknown. Using a combination of computational modeling, biochemical assays, and crosslinking mass spectrometry (XLMS), I modeled the secondary structure of Sec3N and characterized its orientation in relation to exocyst. I also identified multiple phosphorylation sites on Sec3N that were not previously known in the literature. I hypothesized that phosphorylation of Sec3 induces a conformational change which plays an inhibitory role in its function. By showing that Sec3 structure and function can be regulated by phosphorylation, I pinpointed a means by which exocyst can be modulated in a temporal and spatial manner. These findings will help guide future studies in which the specific kinases and phosphatases that play a role in exocytic function will be elucidated. Furthermore, these findings will provide insight into future structural studies on Sec3N and how its conformation plays a role in SNARE complex assembly.