The Yeast Saccharomyces cerevisiae Is Not an Efficient Tool for in Vivo Studies of Plant Vacuolar Sorting Receptors

VSRPS-1 Does Not Cause Significant Accumulation of aleu-GFP in the Vacuole. (A) The Δvps10 mutant containing the aleu-GFP construct was transformed with VPS10 or VSRPS-1 under the control of a GAL promoter. Cells were visualized by fluorescence microscopy during the exponential growth phase, after 24 h of induction in galactose medium at 30°C. Images of GFP fluorescence, of FM4-64 staining of vacuolar membranes, and of intact cells by differential interference contrast (DIC) are shown. (B) Sec-GFP, aleu-GFP, or GFP-Chi fusion proteins were coexpressed with a plant VSR or Vps10p in the Δvps10 mutant using a GAL promoter, as indicated. Equal amounts of each transformant were spotted on inducing galactose medium plates and covered with a nitrocellulose filter. After 24 h at 30°C, the filters were removed and the secreted GFP constructs were detected by immunostaining. (Detailed materials and methods are provided in the supplemental data online). By a similar approach, we then tested whether the family of VSRs identified in Arabidopsis thaliana (AtVSR1, 2, 2′, and 3 to 6) and showing high homology to VSRPS-1 is able to redirect to the vacuole the GFP reporter protein fused to different plant VSDs. The aleu-GFP or the GFP-Chi was expressed alone or together with each putative Arabidopsis VSR in the Δvps10 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 Δvps10 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 Δvps10 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 Δvps10 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 Δvps10 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. VSRPS-1 Is Rapidly Degraded in Yeast Cells, Does Not Travel through the PVC, and Is Not Associated with Golgi or Endosomal Compartments. (A) The HA-tagged VSRPS-1 and HA-tagged Vps10p were expressed in the Δvps10 mutant. After 24 h of induction, protein synthesis was blocked by the addition of CHX. At the indicated times, cells were harvested and whole cell extracts prepared and subjected to immunoblot analysis. Proteins were detected by monoclonal HA antibodies. (B) The degradation of VSRPS-1-HA is not prevented in mutants blocking the biosynthetic transport to the vacuole and in vacuolar protease-deficient cells. VSRPS-1-HA was expressed in the Δvps10, sec18-1 ts, Δvsp45, and Δpep4 strains. Protein expression was induced at 30°C or at 37°C for the sec18-1 ts mutant, and cells were then treated with CHX and analyzed as described in the experimental procedure. C is the control of mutant cells transformed with the vector alone; the asterisk indicates the presence of nonspecific bands. (C) Subcellular localization of the plant receptor VSRPS-1-HA in yeast cells. Δvps10 spheroplasts expressing VSRPS-1-HA were gently lysed by osmotic shock. Subcellular fractionation was then performed by differential centrifugation to yield a low-speed pellet (P13), a high-speed pellet (P100), and a supernatant (S100). The amount of VSRPS-1-HA, Pep12p (a PVC marker), and Vph1p (a vacuolar marker) in each of the fractions was assessed by immunoblotting. (Detailed materials and methods are provided in the supplemental data online). The lack of massive retention of aleu-GFP and the rapid degradation of VSRPS-1 indicate that most of the expressed VSRs are not properly sorted in the yeast cell, thus preventing appropriate interaction with ligands. Vps10p mediates the transport of soluble vacuolar proteins by cycling between the TGN and the PVC (Cooper and Stevens, 1996). 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 Δvps10 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). This work was supported by grants from the Swiss National Science Foundation (FN-3100-065191.01 and FN-31-65403).