Evaluation of a yeast system for studying plant Vacuolar Sorting Receptors (VSRs)

The yeast S.cerevisiae is not an efficient tool for in vivo studies of plant vacuolar sorting receptors Plant and yeast vacuoles, the equivalents of mammalian lysosomes, are acidic compartments involved in hydrolytic functions. They are also essential for metabolite storage and for maintaining cytosolic ion and pH homeostasis (Klionsky and Emr, 1990; Wink, 1993). The vacuoles mainly receive proteins and lipids from the biosynthetic and endocytic vesicular transport pathways. Newly synthesized vacuolar proteins transit through the early compartments of the secretory pathway and are actively sorted away from secreted proteins in the trans-Golgi network (TGN) before being delivered via prevacuolar compartments (PVC) to the vacuoles. In contrast to yeast, the plant vacuolar sorting machinery is extremely complex, since some plant cells may have up to three functionally distinct vacuoles: the lytic vacuole, the storage vacuole (Hoh et al., 1995; Paris et al., 1996) and the neutral vacuole (Di Sansebastiano et al, 1998). Transport analysis of soluble vacuolar proteins in plants identified three classes of vacuolar sorting determinants (VSDs), which likely interact with specific vacuolar sorting receptors (VSR). It is assumed that this interaction allows the VSRs to direct their specific ligands to the right vacuole. Indeed, protein transport to the lytic vacuole has been shown to depend on sequence-specific VSDs (ssVSD) and is mediated by clathrin-coated vesicles (CCVs). Transport to the neutral and (seed) storage vacuoles requires the C-terminal VSD (ctVSD) and the structural type VSD (psVSD), respectively (reviewed in Neuhaus and Rogers, 1998). VSRPS-1 (previously named BP-80) is the first vacuolar sorting receptor identified in plants. VSRPS-1 was originally isolated from pea CCVs by its ability to bind to the ssVSD from barley proaleurain in a pH-sensitive manner in an in vitro assay (Kirsch et al., 1994). Since then, several VSRs were cloned from different plant species (Ahmed et al., 1997; Paris et al., 1997; Shimada et al, 1997) and seven homologues (AtVSR1, 2, 2', 3-6) were identified in A. thaliana (Laval et al., 1999, reviewed in Hadlington and Denecke, 2000). The existence of several homologues in one plant species suggests that these VSRs could have different ligand specificities, and might therefore be involved in the different plant vacuolar pathways and/or function at different stages of plant development (Paris and Neuhaus, 2002). Immunogold electron microscopy showed that both pea and A. thaliana VSRs are predominantly localized in the PVCs as well as in the TGN (Paris et al., 1997; Sanderfoot et al., 1998; Hinz et al., 1999; Li et al., 2002). VSRs also partially colocalized with AtPep12p, an homologue of a yeast t-SNARE that resides on PVCs, in A. thaliana roots (da Silva Conceicao et al., 1997; Sanderfoot et al., 1998) as well as in tomato and tobacco cells (Li et al., 2002). In addition, several proteins, such as t-SNARE AtVAM3p (Sato et al., 1997) and the Sec1p-homologue AtVPS45p (Bassham and Raikhel, 1998) that are involved in vesicular transport to the lytic vacuole have been identified by yeast complementation assays. All these findings have suggested that VSRs travelling through the PVC could function like their yeast counterpart Vps10p, which mediates the transport from the TGN to the PVC of several soluble vacuolar hydrolases such as carboxypeptidase Y (CPY, (Johnson et al., 1987)), proteinase A (Klionsky and Emr, 1998) and several misfolded proteins (Hong et al., 1996). This similarity had been further corroborated by our previous work showing that expression of VSRPS-1 in yeast cells leads to an efficient transport to the vacuole of a GFP fused to the petunia aleurain VSD (Humair et al., 2001). This result demonstrated that plant VSRPS-1 is functional in yeast cells and is capable of interacting, like Vps10p, with the yeast trafficking machinery. In the present study, we further investigated the trafficking of plant VSRs in yeast to determine the ligand specificities of the A. thaliana VSR family. We show that the five tested AtVSRs fail to redirect either aleu-GFP or GFP-Chi to the yeast vacuole. Surprisingly, we were also unable to detect a significant accumulation of aleu-GFP in the vacuole in the presence of VSRPS-1, in contrast to our previous results. Further investigation clearly demonstrated that VSRPS-1 is in fact rapidly degraded and does not reach the PVC in ?vps10 cells. We conclude that plant VSRs do not properly traffic in yeast cells, and therefore, that the yeast trafficking machinery is not a suitable system to study plant vacuolar protein interactions.

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