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© 2005 American Society of Plant Biologists
The Yeast Saccharomyces cerevisiae Is Not an Efficient Tool for in Vivo Studies of Plant Vacuolar Sorting Receptors
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vps10 mutant strain. The GFP retained in the yeast vacuole by AtVSRs was then detected by fluorescence microscopy. None of the five tested AtVSRs significantly retains intracellularly either GFP construct (data not shown). More surprisingly, we were unable to demonstrate the function of VSRPS-1 as a sorting receptor in yeast as described before (Humair et al., 2001
). When compared with Vps10p, no significant accumulation of aleu-GFP in the vacuole was observed in vps10 cells expressing VSRPS-1 (Figure 1A). How do we explain these new confocal observations? Our previous conclusion of a positive assay was made by comparison with the mutant yeast expressing the GFP reporter alone and was based both (1) on the intensity of the vacuolar signal obtained upon expression of the plant VSR and (2) on the number of cells showing fluorescent labeling (up to 60%). Despite the fact that the vacuolar signal was often variable in the negative controls and was on average much weaker in vps10 cells expressing VSRPS-1 than in the wild-type strain, the retention of aleu-GFP by VSRPS-1 was confirmed by immunoblot analysis (Figure 5C in Humair et al., 2001). In this study, we used different growth conditions and observed GFP fluorescence during the late-exponential phase of cell growth, which gave a more homogeneous vacuolar signal in wild-type cells and is more physiologically relevant for studying protein trafficking (Figure 1A; almost 100% of the cells consistently display a vacuolar GFP signal). It is possible that the growth conditions described by Humair et al. (2001), where the fluorescence detection was performed on cells in the late-stationary phase (OD600 between 5 and 10), are more stressful, leading to an accumulation of aleu-GFP in the vacuole in the presence of VSRPS-1. Nevertheless, we doubt that growth conditions alone can explain the observed differences because in the previous study, we had tested cell growth in the late-exponential phase, and in this study, we tested cells in the late-stationary phase. In both cases, there was no influence of growth conditions on the respective confocal observations (data not shown). It is also noteworthy that we previously observed the efficiency of the assay to be higher in less manipulated cells. This observation suggests that VSRPS-1 requires some trafficking components associated with the Vps10p pathway that are quickly downregulated in the yeast mutant strain. The fact that we used the same strain over the length of this study may additionally indicate that this effect is not reversible.
Because the fluorescence background of the aleu-GFP reporter observed in the vacuole of the vps10 mutant was problematic in drawing conclusions, we set up an alternative straightforward approach to demonstrate a functional interaction between the plant VSRs with our different VSD-GFP constructs. In this approach, we took advantage of the fact that most soluble yeast vacuolar proteins are secreted by default in the absence of functional Vps10p. Thus, instead of detecting the GFP that is retained intracellularly by plant VSRs, we determined the amount of secreted GFP binding to a nitrocellulose filter in a colony blot assay (Figure 1B). As expected, a large amount of secreted Sec-GFP and aleu-GFP was detected in the absence of Vps10p. The expression of VSRPS-1 in the vps10 mutant did not lead to a detectable reduction of secreted aleu-GFP compared with cells expressing Sec-GFP or to the control expressing Vps10p. In addition, we also showed that expression of any of the five tested Arabidopsis VSRs (AtVSR1 to 3, 5, and 6) did not significantly prevent the secretion of either aleu-GFP or GFP-Chi (Figure 1B).
Unfortunately, we have no information on the ratio of secreted/retained aleu-GFP when VSRPS-1 is expressed in yeast. We previously estimated that VSRPS-1 has the potential to retain 
30% of aleu-GFP when compared with the wild-type strain (Figure 5C in Humair et al., 2001). It is likely that the colony blot assay, which is designed to detect massive changes in secretion, does not detect such a small variation. Therefore, our failure to detect a decreased secretion of aleu-GFP is not in contradiction with the previously reported quantitative data (Humair et al., 2001) but indicates that the efficiency described before is likely to represent the maximum for this yeast assay. To conclude, our original aim to improve and widely use yeast in a simple quantitative assay for in vivo studies of plant vacuolar receptors was overly optimistic.
To determine the stability and localization of VSRPS-1 in yeast cells, we next expressed a hemagglutinin (HA) epitope–tagged VSRPS-1 (VSRPS-1-HA) under the control of a galactose-inducible promoter (GAL) in the vps10 strain. Cells were grown to early exponential phase at 30°C, and after 24 h of induction in the presence of galactose, cells were treated with cycloheximide (CHX) to block protein synthesis. An equivalent amount of cells was harvested after 0, 15, 30, and 60 min of CHX treatment and subjected to protein gel blot analysis. In contrast with Vps10p-HA, which was not degraded after several hours of CHX treatment, overexpressed VSRPS-1-HA was rapidly degraded (half-life <15 min) (Figure 2A). A similar rate of degradation was also observed for an untagged version of VSRPS-1 using specific monoclonal antibodies against VSRs (data not shown), thus ruling out a negative effect of the HA tag on VSRPS-1 stability. Misfolded proteins that leave the endoplasmic reticulum (ER) or mutated proteins that are not recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) are usually delivered to the vacuole where they are rapidly degraded. This type of degradation is prevented either in vacuolar protease-deficient cells or in mutants blocking protein transport to the vacuole. Indeed, we found that the degradation of VSRPS-1-HA was completely blocked in the sec18-1 (yeast NSF) mutant (Figure 2B), which prevents the fusion of ER-derived vesicles with the cis-Golgi, indicating that VSRPS-1 enters the secretory pathway and is able to reach the cis-Golgi. By contrast, degradation of VSRPS-1-HA still occurred in the vps45 mutant, which blocks transport from the Golgi to the PVC, and in the pep4 mutant, which reduces the activities of major vacuolar proteases (Figure 2B), meaning that the receptor's degradation and its inability to reach the PVC are linked. Consistent with this idea, we also found that VSRPS-1-HA did not sediment with the PVC Pep12p marker (P100) in a differential centrifugation experiment but instead accumulated in larger compartments (P13), including the ER, vacuole, and plasma membrane (Figure 2C). Interestingly, we detected some signal for VSRPS-1-HA in the high-speed pellet (P100) containing the Golgi and PVC in spite of the very short half-life of the protein. This might corroborate our previous immunolocalization results in wild-type cells showing a partial colocalization of VSRPS-1 with its yeast counterpart Vps10p (Figure 6 in Humair et al., 2001) and thus suggests that a small fraction of VSRPS-1 is able to reach the PVC and to be active.
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). The cytoplasmic domain of Vps10p plays a critical role in this transport cycle because it harbors multiple signals that are necessary for the sorting and the stability of the receptor. This was demonstrated by the finding that Vps10p mutants lacking the cytoplasmic tail domain or with mutated residues travel to the PVC before being rapidly degraded in the vacuole (Cooper and Stevens, 1996). The cytosolic tail domain of VSRPS-1 also appears important in recycling to the TGN because a truncated form lacking the cytosolic domain is substantially less stable than the intact receptor in tobacco protoplasts (Jiang and Rogers, 1998). However, the cytosolic domains of VSRPS-1 and Vps10p display no sequence homology, except for a shared Tyr motif, and differ in length (38 versus 164 residues). This suggests that the cytosolic domain of VSRPS-1 lacks most sorting signals to selectively interact with the yeast trafficking machinery and that VSRPS-1 is unable to cycle efficiently between the TGN and the PVC. Because the localization of certain proteins may also depend on the length and the amino acid composition of their transmembrane domain (TMD) (Sato et al., 1996; Rayner and Pelham, 1997), the mislocalization of VSRPS-1 could be due either to its longer TMD (23 versus 17 residues for Vps10p) or to its sequence. Thus, to improve the stability and sorting of VSRPS-1, we constructed a hybrid protein in which the VSRPS-1 luminal domain was fused to the Vps10p-HA TMD and cytosolic domains. Although this hybrid protein was less rapidly degraded than VSRPS-1, it did not redirect the aleu-GFP to the vacuole as judged by fluorescence and still sedimented in lower density fractions than Golgi and endosomal membranes (data not shown). Therefore, it is possible that the luminal domain of VSRPS-1 is responsible for its instability. This would be in agreement with previous observations showing that luminal domains of membrane proteins can affect their stability and localization. For instance, various deletions in the luminal domain of Vps10p result in either instability or mislocalization (Jorgensen et al., 1999). In addition, increasing evidence indicates that proteins with minor conformational defects that transit through the Golgi are retrieved to the ER to be either properly refolded or degraded by proteasomes (Caldwell et al., 2001; Vashist et al., 2001). Therefore, it is possible that the majority of VSRPS-1 does not pass the quality control in the cis-Golgi compartment and is transported back to the ER to be dislocated to the cytosol and then degraded by the proteasomes.
Our original work aimed at showing an in vivo interaction between VSRPS-1 and a vacuolar protein precursor, which could not yet be shown in plants. Meanwhile, it has recently been reported that a homozygous knockout of AtVSR1 causes mutant plants to missort a subset of the seed storage proteins to the cell wall, without effect on other tissues (Shimada et al., 2003). A soluble, ER-retained form of the pumpkin VSR PV72 specifically causes the accumulation in the ER of a vacuolar protein precursor (Watanabe et al., 2004). These two results indicate that VSRs can indeed act in planta as sorting receptors.
In summary, we showed that the majority of VSRPS-1-HA expressed in the yeast vps10 mutant strain is rapidly degraded, most likely because of inefficient transport and/or retention to the PVC. This demonstrates that yeast is not well suited for study of the in vivo function of plant VSRs and supports the idea that "plant cells are not just green yeast" (Bassham and Raikhel, 2000).
Acknowledgments
This work was supported by grants from the Swiss National Science Foundation (FN-3100-065191.01 and FN-31-65403).
Footnotes
Online version contains Web-only data. 
REFERENCES
Bassham, D.C., and Raikhel, N.V. (2000). Plant cells are not just green yeast. Plant Physiol. 122, 999–1001.
Caldwell, S.R., Hill, K.J., and Cooper, A.A. (2001). Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi. J. Biol. Chem. 276, 23296–23303.
Cooper, A.A., and Stevens, T.H. (1996). Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J. Cell Biol. 133, 529–541.
Hong, E., Davidson, A.R., and Kaiser, C.A. (1996). A pathway for targeting soluble misfolded proteins to the yeast vacuole. J. Cell Biol. 135, 623–633.
Humair, D., Hernandez Felipe, D., Neuhaus, J.M., and Paris, N. (2001). Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor. Plant Cell 13, 781–792.
Jiang, L., and Rogers, J.C. (1998). Integral membrane protein sorting to vacuoles in plant cells: Evidence for two pathways. J. Cell Biol. 143, 1183–1199.
Jorgensen, M.U., Emr, S.D., and Winther, J.R. (1999). Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae. Eur. J. Biochem. 260, 461–469.[Web of Science][Medline]
Rayner, J.C., and Pelham, H.R. (1997). Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast. EMBO J. 16, 1832–1841.[CrossRef][Web of Science][Medline]
Sato, M., Sato, K., and Nakano, A. (1996). Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain. J. Cell Biol. 134, 279–293.
Shimada, T., Fuji, K., Tamura, K., Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2003). Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 100, 16095–16100.
Vashist, S., Kim, W., Belden, W.J., Spear, E.D., Barlowe, C., and Ng, D.T. (2001). Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J. Cell Biol. 155, 355–368.
Watanabe, E., Shimada, T., Tamura, K., Matsushima, R., Koumoto, Y., Nishimura, M., and Hara-Nishimura, I. (2004). An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves. Plant Cell Physiol. 45, 9–17.
Wooding, S., and Pelham, H.R. (1998). The dynamics of Golgi protein traffic visualized in living yeast cells. Mol. Biol. Cell 9, 2667–2680.
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