Abstract
We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) 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 RNA interference 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 carboxy-terminal vacuolar sorting determinants 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 Sec1/Munc18 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, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.
Highlights
Departamento de Genetica Molecular de Plantas, Centro Nacional de Biotecnologıa, Consejo Superior de Investigaciones Cientıficas, E–28049 Madrid, Spain (J.Z., E.R.); and Department of Genetics, Development, and Cell Biology and Plant Sciences Institute, Iowa State University, Ames, Iowa 50011 (D.C.B.)
We report a functional characterization of AtVPS45, a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex
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 Sec1/Munc18 regulator
Summary
AtVPS45 is encoded by a single gene containing 13 exons on chromosome I. We observed a clear acceleration of senescence in siVPS45 lines relative to wild-type lines (compare the total chlorosis after 4 d in leaves from the silenced lines with the presence of chlorophyll in wild-type leaves after 6 d; Fig. 2C), similar to that reported for vti mutants (Surpin et al, 2003), supporting a role for AtVPS45 in nutrient recycling We conclude from these results that AtVPS45 is likely involved in vesicle trafficking to the vacuole and that reduced levels of this protein result in defective vacuole formation and function. These data suggest that AtVPS45 silencing does not lead to general secretion of endogenous vacuolar proteins but affects trafficking of the ctVSD-containing proteins like VAC2, GFP-CHI, and AtAleurain
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