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.
Highlights
Synthesized organellar proteins are delivered to their respective organelles by a complex mechanism of transport
binding protein (BiP) and calreticulin were detected at their expected positions, and clathrin was detected as high molecular weight forms, as previously described (Kim et al, 2001)
In this study, we demonstrated that AtVSR1 forms high molecular weight complexes through homomeric interaction in vivo
Summary
Synthesized organellar proteins are delivered to their respective organelles by a complex mechanism of transport. In the TGN, they are involved in sorting of vacuolar proteins containing a vacuolar sorting motif, NPIR, for packaging into clathrin-coated vesicles (CCVs) In support of this theory, it was shown that in vitro, BP80/VSR binds to the N-terminal propeptide sorting signal, the NPIR motif (Kirsch et al, 1994, 1996; Ahmed et al, 1997, 2000). Overexpression of the ER-localized luminal domain of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves (Watanabe et al, 2004). Storage proteins were secreted into the apoplastic space of Arabidopsis seeds In this case, the sorting signal recognized by AtVSR1 may be different from the NPIR motif found in proteins destined to the central vacuole
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