In vitro Reconstitution of Rab-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog, Sro7 [Cell Biology]

November 17th, 2014 by Rossi, G., Watson, K., Demonch, M., Temple, B., Brennwald, P.

Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.