Homomeric Interaction of AtVSR1 Is Essential for Its Function as a Vacuolar Sorting Receptor

Abstract

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

“…GFP:SYP61 localized SYP61-positive compartments (see Supplemental Figure 4 online). In both wild-type and mag1-1 protoplasts, the majority (>80%) of VSR1:HA closely colocalized with GFP:SYP21 and a small proportion (<20%) of VSR1:HA overlapped with GFP:SYP61 ( Figures 5A and 5C), indicating that VSR1 primarily localizes to the PVC in both wild-type and mag1-1 protoplasts as reported previously (Li et al, 2002;Tse et al, 2004;Kim et al, 2005Kim et al, , 2010. This result implied that, if VSRs recycle proteins for vacuolar trafficking, they would recycle from the PVC to the TGN.…”

“…VSRs play a crucial role in sorting soluble proteins at the TGN and recycle from the PVC to the TGN (Li et al, 2002;Tse et al, 2004;Kim et al, 2010), although a recent study proposed that VSRs recycle from the TGN to the ER, where they are involved in sorting vacuolar proteins (Niemes et al, 2010a). Arabidopsis VPS29 is a component of the retromer complex (Oliviusson et al, 2006;Shimada et al, 2006;Jaillais et al, 2007), which is thought to be responsible for recycling VSRs (Oliviusson et al, 2006).…”

“…Accordingly, we tested whether VSR1:HA recycles from the PVC to the TGN by examining the localization of VSR1:HA in the presence of latrunculin B (LatB), a chemical compound that inhibits actin filament assembly. Previous studies demonstrated that LatB inhibits anterograde trafficking of VSR1 from the TGN to the PVC but allows retrograde trafficking from the PVC to the TGN, which in turn results in the accumulation of VSR1 at the TGN (Kim et al, 2005;Kim et al, 2010). VSR1:HA was cotransformed with GFP:SYP61 or GFP:SYP21.…”

“…Seven VSRs localize primarily to the PVC (Tse et al, 2004). To determine the localization of a specific VSR isoform, we used VSR1 tagged with HA (VSR1:HA) (Kim et al, 2010). VSR1:HA was cotransformed into protoplasts from wild-type or mag1-1 mutant plants along with GFP:SYP61 or GFP:SYP21, and the localization of these proteins was examined directly or by immunohistochemistry using anti-HA antibody.…”

“…Therefore, to identify the intermediate compartments in which these proteins were trapped, we compared the localization of Spo:GFP or AALP:GFP with that of organelle markers. We used KAM1(DC):mRFP, a chimeric fusion between Katamari1 (KAM1) harboring a C-terminal deletion and monomeric red fluorescent protein (mRFP); this fusion localizes to the cis-Golgi apparatus (Tamura et al, 2005;Lee et al, 2011), SYP61, a t-SNARE localized to the TGN (Sanderfoot et al, 2001;Uemura et al, 2004), and HA-tagged PEP12p, a t-SNARE localized to the PVC (Sanderfoot et al, 1998;Kim et al, 2010), as organelle marker proteins. KAM1(DC):mRFP or PEP12p:HA were cotransformed into mag1-1 protoplasts together with Spo:GFP or AALP: GFP and their localization was examined directly or by immunostaining with an anti-HA antibody or an anti-SYP61 antibody.…”