AtVPS45 is a positive regulator of the SYP41/SYP61/VTI12 SNARE complex involved in trafficking of vacuolar cargo

Abstract

We report a functional characterization of AtVPS45, a protein from the Sec1/Munc18 (SM) family that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNAi lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with C-terminal vacuolar sorting determinants (ctVSD) was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate SM regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45 silenced plants we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors (VSRs), which are required for sorting of ctVSD-containing cargo. We propose that AtVPS45 is essential for recycling of the VSRs back to the TGN, and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.