The Nutrient Stress-induced Small GTPase Rab5 Contributes to the Activation of Vesicle Trafficking and Vacuolar Activity*

Background: How the function of Rab5 isoforms is regulated remains unclear. Results: The third Rab5 isoform, Ypt53, is up-regulated significantly under nutrient stress. Conclusion: The up-regulated Ypt53 and the constitutive Vps21 are crucial for vesicle transport and vacuolar hydrolase activity, and they prevent ROS accumulation and mitochondrial dysfunction. Significance: The function of three Rab5 isoforms may be regulated differently to adapt to environmental changes. Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.

Eukaryotic cells are compartmentalized into multiple distinct membrane-bound organelles, and elaborate systems are required to transfer proteins and lipids between these compartments. Members of the Rab family of small GTPases are major regulators of macromolecular traffic in the secretory and endocytic pathways (1)(2)(3)(4). Rab GTPases function as molecular switches that alternate between two conformational states: the GTP-bound "on" form and the GDP-bound "off" form. Conversion of the GDP-bound Rab into the GTP-bound form is mediated through the exchange of GDP for GTP, which is catalyzed by a guanine nucleotide exchange factor. The GTP-bound "active" Rabs bind effector proteins and complexes that execute diverse functions in membrane trafficking. The active Rabs are converted back to the GDP-bound "inactive" form by hydrolysis of GTP, which is stimulated by a GTPase-activating protein.
Rabs are anchored to membranes through C-terminal prenyl anchors. In their inactive GDP-bound state, Rabs can be extracted from membranes and relocated by the cytosolic chaperone GDP dissociation inhibitor, which binds the prenyl groups of Rabs and delivers them to their target membrane.
In both yeast and mammals, there are three Rab5 isoforms (Vps21, Ypt52, and Ypt53 in yeast and Rab5a, Rab5b, and Rab5c in mammals) that reside on endosomes. Rab5 regulates the transport of proteins from early to late endosomes and then to terminal lysosomes (vacuoles) by controlling vesicle budding, uncoating, motility, tethering, and fusion (5)(6)(7)(8)(9)(10). More recently, Rab5 has been shown to be necessary for the biogenesis of the endolysosomal system in vivo (11). The multitasking ability of Rab GTPases is underscored by their spatiotemporal coordination of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases, and various regulators of membrane trafficking. For example, the Vps21-mediated tethering activity may be promoted by the class C core vacuole/ endosome tethering complex and also regulated by upstream activators, inhibitors, and effectors (9,(12)(13)(14)(15). However, in contrast to the accumulated knowledge on the catalytic activity of Vps21, much less is known about the mechanisms by which the functions of other Rab5 isoforms are regulated (16,17). In particular, our understanding of the molecular mechanisms controlling the expression of Rab proteins is still limited, although their altered expression has now been analyzed in cells of the immune system (18).
In a previous study, we demonstrated that the activity of Ypt52 is regulated negatively by Roy1, a non-Skp1-Cul1-F-boxtype F-box protein. In this study, we demonstrate that another isoform of Rab5 in Saccharomyces cerevisiae, Ypt53, is up-regulated significantly under nutrient stress conditions. We found that constitutively expressed Vps21 plays a major role in vesicle trafficking under normal conditions and in the activation of vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, Ypt53 is up-regulated significantly and functions redundantly with Vps21 to facilitate intracellular trafficking. Moreover, Ypt53 and Vps21 are both critical to prevent the accumulation of reactive oxygen species ROS 3 and to maintain mitochondrial respiration. Therefore, we propose that Ypt53 functions as a backup factor that confers robustness against nutrient stress by expanding Rab5 signaling.
Plasmids-The plasmid encoding Candida glabrata HIS3 gene (pUC19-CgHIS3) was provided by the National Bio-Resource Project of MEXT, Japan. The plasmid encoding YPT53 under the control of the VPS21 promoter was constructed as follows. The VPS21 promoter (1000 bp) and the open reading frame of the YPT53 gene were amplified by PCR and cloned into pRS316. The plasmid encoding GFP-Atg8 was constructed as follows. The promoter region of ATG8 (1000 bp), the gene for enhanced GFP (derived from pTYE481, a gift from T. Yoshihisa, University of Hyogo, Hyogo, Japan), and the open reading frame and the terminator (1000 bp) of ATG8 were inserted into pRS313 to give P ATG8 -GFP-ATG8-T ATG8 . A pRS316-based plasmid encoding P ATG8 -GFP-ATG8-T ATG8 was also used (a gift from H. Nakatogawa and Y. Ohsumi, Tokyo Institute of Technology, Kanagawa, Japan). All primer sequences are available upon request.
RT-PCR-Isolation of total RNA and cDNA synthesis were performed using TriPure isolation reagents (Roche) and Rever-Tra Ace (Toyobo), respectively. All primer sequences are available upon request.
Antibodies and Immunoblot Analysis-Polyclonal antibodies to Ypt53 were generated in a rabbit by standard procedures with recombinant Ypt53 as an antigen. The anti-Vps21 and Ypt52 antibodies have been described previously (19). The mouse mAb to Pgk1 was purchased from Invitrogen. The rabbit polyclonal antibody to CPY was a gift from T. Endo (Nagoya University, Nagoya, Japan). The rabbit polyclonal antibody to Ape1p was a gift from H. Nakatogawa and Y. Ohsumi (Tokyo Institute of Technology, Kanagawa, Japan).
The concentrations of Ypt53 and Vps21 in cells were estimated as follows. His 6 -tagged Ypt53 and Vps21 were expressed from pET30a (Novagen) in JM109 (DE3) and purified by nickelnitrilotriacetic acid-agarose (Wako). These purified proteins were used as a standard to calculate the amount of Ypt53 and Vps21 in whole cell lysate by Western blotting with anti-Ypt53 and anti-Vps21 antibodies. The amount of each protein in 1 A 600 equivalent of cells was calculated.

Expression of Ypt53 Is Induced under Nutrient Stress Conditions-Previous
reports have demonstrated that ypt53⌬ cells do not show any clear phenotypes under normal growth conditions (5,15). Other reports implied that the expression of Ypt53 is lower than that of Vps21 and Ypt52 (24). During our analysis of endogenous Rab5 proteins using specific antibodies, we found a significant increase in Ypt53 over the time when cells were grown to post-log phase in glucose-containing medium (Fig. 1, A and B, lanes 2-5), although it was almost undetectable during log phase (lane 1). Yeast cells preferentially ferment glucose to produce ATP but can also metabolize a non-fermentable carbon source, such as glycerol or ethanol, for ATP production. When cells exhaust glucose in medium, they start to use ethanol that accumulates from the prior fermentation of glucose (25). This conversion is termed the "diauxic shift." The up-regulation of Ypt53 in glucose-grown cells is most likely triggered by the diauxic shift because we observed a dramatic increase in the expression of Ypt53, but not of Vps21 and Ypt52, when the carbon source was shifted from glucose to glycerol or ethanol (Fig. 1C). The induction of Ypt53 in glycerol-grown cells appeared to occur at the transcriptional level (Fig. 1D). In addition to the induction of Ypt53 at the diauxic shift, we also noted that the level of Ypt53 continued to increase over time, even after cells entered the stationary phase (Fig. 1B, Ͼ48 h). At the stationary phase, cells exhaust available carbon sources and are exposed to nutrient stress (25). Indeed, the induction of Ypt53 at the stationary phase was dependent on Gis1 (Fig. 1E), a transcription factor that is known to regulate genes during nutrient limitation (26). Together, these results suggest that the expression of Ypt53 is induced under nutrient stress conditions. As far as we know, this is the first instance of Rab5 GTPase expression being regulated by nutrient status.
Role of Ypt53 and Vps21 in Vesicle Trafficking at Post-log Phase-To investigate the role of the induced Ypt53 in vesicle trafficking, we monitored the maturation of carboxypeptidase Y (CPY), a soluble vacuolar marker protein (27). In the endoplasmic reticulum (ER), a prepro form of CPY undergoes proteolytic cleavage and addition of N-linked oligosaccharide chains to generate a 67-kDa ER form, "p1CPY." In the Golgi complex, p1CPY is converted to a 69-kDa form, "p2CPY," by addition of mannose to the N-linked glycans. After reaching the vacuole, p2CPY becomes a 61-kDa mature form, "mCPY." As shown in Fig. 2A, when wild-type cells were grown in glucosecontaining medium to early log phase (A 600 , ϳ0.5), CPY was detected as the mature form (lane 1, ␣-CPY blot). Similarly, in ypt53⌬ cells, CPY was transported normally to the vacuole ( Fig.  2A, lane 2). In contrast, in vps21⌬ cells, the total amount of CPY decreased, and the p1 and p2 forms accumulated ( Fig. 2A, lane   3). This is consistent with previous reports that a vast majority of CPY is missorted into the extracellular space upon deletion of VPS21. A similar result was obtained for vps21⌬ypt53⌬ cells ( Fig. 2A, lane 4). When cells were grown to post-log phase (Ͼ60 h of culture), CPY was still detected as a mature form in wildtype and ypt53⌬ cells ( Fig. 2A, lanes 5 and 6). Surprisingly, a significant portion of CPY was also delivered to the vacuole, even in vps21⌬ cells ( Fig. 2A, lane 7). Additional deletion of YPT53 in vps21⌬ cells prevented the transport of CPY to the vacuole ( Fig. 2A, lane 8). Similar results were obtained when cells were grown in glycerol-containing medium ( Fig. 2A, lanes  9 -16). These results suggest that the induced Ypt53 during post-log phase can contribute to Golgi-vacuole protein transport under nutrient stress conditions. We next investigated the relative concentrations of Ypt53 and Vps21 during post-log phase. We found that the amount of Ypt53 was 10-to 15-fold less than that of Vps21. The concentration of Ypt53 was estimated to be ϳ5 ng/1.0 A 600 cells, and that of Vps21 was ϳ50 ng/1.0 A 600 cells. This difference is in line with the fact that the loss of Ypt53 did not cause any defect in CPY transport during post-log phase ( Fig. 2A, lanes 6 and  14), whereas the loss of Vps21 caused a slight defect in this event (lanes 7 and 15). Together, although the contribution of Vps21 is still larger than that of Ypt53, these two Rab5 GTPases may function redundantly and support the vacuolar transport of CPY during post-log phase.
The idea for the functional redundancy between Vps21 and Ypt53 can be supported by the fact that they share the highest FIGURE 1. Ypt53 is up-regulated during post-log phase. A, growth curve of the wild-type strain. Wild-type cells were grown in YPglucose to mid-log phase and diluted to a concentration of 0.2 A 600 /ml. Cells were grown further, and the concentration of cells was measured at the indicated time points. B, cells were grown in YPglucose or YPglycerol to mid-log phase and diluted to a concentration of 0.2 A 600 /ml in the same medium. At the indicated time points, cells were collected, and cell extract was prepared. The level of Ypt53 was assessed by Western blotting with specific antibodies. Pgk1 served as a loading control. C, cells were grown in YPglucose to mid-log phase and transferred to YPglucose, YPglycerol, or YPethanol for 2 h. The levels of Ypt53, Vps21, and Ypt52 were measured by Western blotting with specific antibodies. Pgk1 served as a loading control. D, cells were grown in YPglucose to mid-log phase and transferred to YPglucose or YPglycerol for 2 h. The levels of mRNA for YPT53, VPS21, and YPT52 were measured by quantitative RT-PCR. ACT1 served as an internal control. E, wild-type and gis1⌬ cells were grown to post-log phase (ϳ3 days). Cells were collected, and the level of Ypt53 was assessed by Western blotting with specific antibodies. Pgk1 served as a loading control.

Rab5 Isoform Regulation under Nutrient Stress Conditions
sequence similarity in three Rab5 isoforms in yeast (5). Vps21 and Ypt53 share ϳ57% identity in amino acid sequence, whereas Vps21 and Ypt52 share ϳ48% identity, and Ypt52 and Ypt53 share ϳ53% identity. Moreover, we observed a synthetic growth defect of cells lacking both Ypt53 and Vps21 during post-log phase in glucose-and glycerol-containing medium (Fig. 2B). Finally, when Ypt53 was overexpressed from a plasmid under the control of the VPS21 promoter, it successfully rescued the transport defect of CPY in the vps21⌬ypt52⌬ypt53⌬ triple mutant cells (Fig. 2C). These results suggest that Ypt53 has an overlapping function with Vps21.
Ypt53 Functions Redundantly with Vps21 to Help Strengthen Vacuolar Hydrolase Activity under Nutrient-limited Conditions-Autophagy is a nonspecific degradation process that is highly conserved among eukaryotes (28 -30). Under nutrientlimited conditions, cytosolic double-membrane vesicles emerge and sequester cytosolic proteins and organelles as cargo, and then deliver this cargo to the lysosomes/vacuoles for degradation (28 -30). Because the expression of Ypt53 turned out to be related to the nutrient status of the cell, we wondered whether its expression might also be induced under nitrogen starvation conditions, where vacuolar hydrolase activity is necessary for autophagy. Strikingly, as shown in Fig. 3A, Ypt53 was strongly up-regulated when cells were shifted from rich medium to nitrogen starvation medium. The levels of Vps21 and Ypt52 were unchanged under the same conditions. Because depletion of Vps21 and Ypt53 resulted in a major defect in the delivery of CPY to the vacuole, we reasoned that the up-regulated Ypt53 might contribute to strengthen vacuolar hydrolase activity under nutrient-limited conditions. To investigate this possibility, we monitored the processing of GFP-Atg8. Upon delivery of GFP-Atg8 to the vacuole via the autophagy pathway, Atg8 was degraded rapidly by proteinases in the vacuole, whereas the released GFP remained relatively stable (31,32). In wild-type and ypt53⌬ cells, the amounts of free GFP increased over time after cells were shifted to nitrogen starvation medium (Fig. 3B, lanes 1-8). In contrast, no free GFP was detected in vps21⌬ and vps21⌬ypt53⌬ cells, suggesting a crucial role of Vps21 in the activation of vacuolar hydrolase.
In parallel with GFP-Atg8, we monitored the processing of aminopeptidase 1 (prApe1) (31,33). The precursor form of Ape1 that is synthesized in the cytosol is delivered to the vacuole via the cytosol-to-vacuole targeting pathway under nutrient-rich conditions and through autophagy under nitrogen starvation conditions (34). After entering the vacuole, the N-terminal propeptide of prApe1 is cleaved to generate the mature form (mApe1). The maturation of prApe1 appeared to be normal in ypt53⌬ cells compared with wild-type cells under rich conditions (Fig. 3B, lanes 1 and 5) and under nitrogen starvation conditions (Fig. 3B, lanes 2-4 and 6 -8). However, in vps21⌬ and vps21⌬ypt53⌬ cells, mature Ape1 was almost undetectable, even under rich conditions (Fig. 3B, lanes 9 and 13; see also Fig. 3C, lanes 5 and 7). A defect in Ape1 maturation under nutrient-rich conditions in these mutants may be due to a block in the cytosol-to-vacuole targeting pathway and/or an inefficient cleavage of the Ape1 propeptide in the vacuole by lumenal proteases that were transported into the vacuole inefficiently because of the depletion of Rab5 proteins ( Fig. 2A). When these cells were shifted to nitrogen-starved medium, only a slight amount of mature Ape1 was observed in vps21⌬ cells, and its degree was indistinguishable from that in vps21⌬ypt53⌬ cells (Fig. 3B, lanes 10 -12 and 14-16). These results demonstrate that, in contrast to our initial expectation, the contribution of Ypt53 to facilitating the vacuolar hydrolase activity is only limited, whereas Vps21 plays a major role in its activity, at least during the period shortly after cells were shifted to nitrogen starvation conditions. However, when cells were incubated under nitrogen starvation conditions for an extended period of time (ϳ24 h), we noticed that Ypt53 continued to increase over time (Fig. 3C,  lanes 1-2). Importantly, after prolonged incubation, GFP-Atg8 and prApe1 were processed eventually, even in vps21⌬ cells (Fig. 3C, lane 6), whereas the processing of these marker proteins was reduced strongly in vps21⌬ypt53⌬ cells (Fig. 3C, lane  8). The induction of Ypt53 under nitrogen-starvation condition could also partially rescue the CPY transport defect in vps21⌬ cells (Fig. 3D, compare lanes 6 and 8). The partial rescue of the vps21⌬ phenotype by Ypt53 can be explained by the relative concentration of Vps21 and Ypt53. The amount of Ypt53 and Vps21 proteins under nitrogen starvation conditions was estimated to be ϳ9 ng/1.0 A 600 cells and ϳ45 ng/1.0 A 600 cells, respectively. Although these results suggest that the processing defect of GFP-Atg8 and prApe1 was caused by the incomplete transport of vacuolar proteins, it is still possible that the autophagy pathway is somehow impaired in vps21⌬ypt53⌬ cells. To test this possibility, we first analyzed the autophagic bodies in vacuoles. As shown in Fig. 3E, autophagic body-like granular structures were observed in the vacuole of vps21⌬ypt53⌬ cells after incubation under starvation conditions (Fig. 3E). Interestingly, these structures appeared to be less motile than autophagic bodies in pep4⌬ cells (data not shown). To further analyze the autophagy pathway, we observed the localization of GFP-Atg8.
As shown in Fig. 3F, when wild-type cells were shifted to nitrogen starvation conditions, GFP-Atg8 was transported into the vacuole, and the subsequently released free GFP was diffused throughout the vacuole lumen. In hydrolase-deficient cells such as pep4⌬, it has been well established that autophagic bodies are not broken down and that they cluster together. Indeed, GFP-Atg8 accumulated in the vacuole lumen (32). We then analyzed GFP-Atg8 in vps21⌬ypt53⌬ mutant cells. Intriguingly, we observed multiple granular structures of GFP-Atg8. These structures seemed to be distributed throughout the vacuole and looked different from the staining pattern in pep4⌬ 2) for the indicated times (0, 2, 4, and 6 h). The levels of Ypt53, Vps21, and Ypt52 were analyzed by Western blotting with specific antibodies. Pgk1 served as a loading control. B, cells expressing GFP-Atg8 were incubated in synthetic complete medium to early log phase and transferred to SD-N ϩ 50 mM MES-KOH medium (pH 6.2). Cells were collected at the indicated time points, and the processing of Ape1 and GFP-Atg8 was assessed by Western blotting with anti-Ape1 antibody and anti-GFP antibody, respectively. The precursor and mature forms of Ape1 are labeled prApe1 and mApe1, respectively. The asterisk indicates a nonspecific band. C, Ypt53 and Vps21 function redundantly to activate vacuolar hydrolase after prolonged incubation of cells under nitrogen starvation conditions. Cells expressing GFP-Atg8 were incubated in synthetic complete medium to mid-log phase and transferred to SD-N ϩ 50 mM MES-KOH medium (pH 6.2). After cells were cultured for 24 h, the processing of Ape1 and GFP-Atg8 was analyzed as in B. Pgk1 served as a loading control. The asterisks show nonspecific bands. It should be noted that the amount of mApe1 in wild-type cells during early log phase (B, lane 1) was less than that during mid-log phase (C, lane 1). This is consistent with the observation that the amount of mApe1 under the normal nutrient conditions largely depends on the growth phase. mApe1 accumulates during mid-to late log phase more than during the early log phase (H. Nakatogawa, personal communication). D, the induced Ypt53 partially compensates for the CPY transport defect caused by depletion of Vps21 under nitrogen starvation conditions. Cells were grown in synthetic complete medium, transferred to SD-N ϩ 50 mM MES-KOH medium (pH 6.2), and incubated for a further 24 h. Cell extracts were prepared, and CPY was detected with its specific antibody. The ER form (p1), Golgi form (p2), and vacuole form (m) of CPY are indicated. Pgk1 served as a loading control. E, cells were grown in synthetic complete medium overnight to mid-log phase, washed twice with excess amount of distilled water, and incubated in S-N ϩ 50 mM MES-KOH medium (pH 6.2) supplemented with 1 mM PMSF for 3 h. Nomarski images were captured and analyzed with Axio Vision 4.6. Scale bar ϭ 2 m. F, the indicated cells expressing GFP-Atg8 were first grown in synthetic complete medium overnight to mid-log phase. Cells were then incubated with FM4-64 dye for 1 h. Cells were washed twice with an excess amount of distilled water and incubated in SD-N ϩ 50 mM MES-KOH medium (pH 6.2) for 4 h. Nomarski images (DIC: differential interference contrast), GFP, and FM4-64 images were captured and analyzed with Axio Vision 4.6. Scale bar ϭ 2 m. cells, where GFP-Atg8 accumulates and forms relatively small structures in the vacuole. It should be noted that a vacuolar marker, FM4-64 dye (35), reached the vacuole less efficiently in vps21⌬ypt53⌬ cells, probably because this strain shows a strong defect in endocytosis.
One possible scenario that could explain these observations is that the formation and transport of the autophagosome may be unaffected in vps21⌬ypt53⌬ cells. However, subsequent steps (i.e. the release of the autophagic body from the vacuolar membrane into the lumen) might somehow be impaired. This could be one reason for the lower motility of the structure as well as the processing defect of GFP-Atg8. At the same time, it is still possible that the processing defect of GFP-Atg8 was caused by the defect in the vacuolar hydrolase activity. We believe that these two possibilities can occur simultaneously and not in a mutually exclusive manner. Regardless, these observations should form a basis for further investigation of a potentially direct role of Rab5 in autophagy in yeast.
Nutrient Stress-induced Ypt53 and the Constitutively Expressed Vps21 Function Together to Prevent the Accumulation of ROS and to Maintain Mitochondrial Respiratory Activity-Previous studies have suggested that defects in autophagy cause the accumulation of ROS (22, 36 -38). The reason for ROS accumulation may well be explained by the imbalance of mitochondrial respiratory enzymes, inefficient expression of ROS scavenger proteins, and/or defects in the autophagic degradation of mitochondria (mitophagy) (22,38). Therefore, we asked whether depletion of Vps21 and Yp53 might cause the enhanced carbonylation, a non-enzymatic protein modification catalyzed by ROS (39). As shown in Fig. 4, when cells were grown to post-log phase (Ͼ70 h), carbonylated proteins accumulated slightly in vps21⌬ cells (lane 3), and a significant amount of them accumulated in vps21⌬ypt53⌬ cells (lane 4) compared with wild-type and ypt53⌬ cells (lanes 1 and 2). This result supports our idea that Vps21 and Ypt53 have an overlap-ping function in decreasing ROS under nutrient-limited conditions. ROS accumulation and/or defects in vacuolar activities are known to lead to the loss of mitochondrial DNA and result in a respiratory-deficient phenotype (40,41). To test whether deletion of VPS21 and YPT53 might cause a respiratory-deficient (petite) phenotype under nutrient-limited conditions, we took advantage of the ade2-1 mutation of W303 strains. Yeast cells with mutations in the ADE2 gene (W303 strains) accumulate a red pigment because of the disruption of the adenine biosynthetic pathway on YPglucose plates. However, yeast cells without functioning mitochondria like "petite" do not reach this step in the adenine synthesis pathway and, thus, form white colonies (42,43). When cells were cultured under nitrogen starvation conditions for 2 days, only 4ϳ8% of wild-type, ypt53⌬, and vps21⌬ cells formed white colonies (Fig. 5, A and B). In contrast, Ͼ30% of vps21⌬ypt53⌬ cells formed white colonies. White colonies were streaked on YP glycerol plates and were confirmed to be respiratory-deficient (Fig. 5C). On the basis of these genetic and biochemical studies, we propose that the constitutively expressed Vps21 and the nutrient stress-induced Ypt53 function together to maintain vacuolar hydrolase activities, which prevent the accumulation of ROS and maintain mitochondrial respiration, demonstrating the physiological importance of the up-regulation of Ypt53 (Fig. 6).

DISCUSSION
In this study, we discovered an additional layer of regulation of Rab5 signaling in yeast in which an isoform of Rab5, Ypt53, is up-regulated under nutrient stress conditions. To our knowledge, this is the first example of Rab5 GTPase expression being controlled by nutrient status. Under nutrient-rich conditions, the constitutively expressed Vps21 plays a critical role in vesicle transport and in facilitating the vacuolar hydrolase activities. However, when cells are shifted to nutrient-limited conditions, the constitutive Vps21 and the induced Ypt53 function redundantly and facilitate these activities. Moreover, the function of both Vps21 and Ypt53 is essential for preventing the accumulation of ROS and for maintaining mitochondrial respiratory activity under nutrient-limited conditions (Fig. 6). Therefore, we propose that the up-regulated Ypt53 functions in a "stagespecific" manner that is dependent on nutrient status and helps strengthen net Rab5 activity, thereby allowing cells to adapt to environmental changes.
Cells have evolved various mechanisms to adapt to different nutrient conditions. Autophagy is one such mechanism that sequestrates cytosolic cargo for degradation in the vacuole. Autophagy is not only a housekeeping process, but it is also activated under nutrient-limited conditions to provide supplementary reserves for the starving cells (44). However, during nutrient starvation, the influx of cargo (proteins and lipids) into the vacuole by autophagy exerts increasing pressure on the degradation systems in the vacuole. To overcome this pressure, the hydrolytic capacity of the vacuole should also be increased under nutrient-limited conditions. The up-regulated Ypt53 may well contribute to the increase in vacuolar hydrolase activity, most likely by facilitating the transport of vacuolar enzymes. In addition, we have shown that the up-regulation of Ypt53 is mediated, at least in part, by the transcription factor Gis1 (Fig.  1E). Gis1 is known to be negatively regulated by Tor1 (target of rapamycin) (45). Therefore, we propose that, under nutrientlimited conditions, Tor1 is inactivated and that the activated Gis1 may up-regulate Ypt53. In this regard, it is interesting to note that Rab5 proteins both in yeast and mammals regulate the activity and localization of TORC1 (16). One attractive hypothesis is that the up-regulated Rab5 signaling under nutrient limited conditions functions as a positive feedback mechanism that modulates TORC1 activity and its downstream signaling cascade.
Both Vps21 and Ypt53 have been shown to bind to Vps8 (12,13), which is one of the effector proteins that associate with class C core vacuole/endosome tethering, suggesting that Vps21 and Ypt53 may share downstream effector proteins. Consistent with this notion, the up-regulated Ypt53 could partially rescue the vps21⌬ phenotype (i.e. the intracellular transport and the activation of vacuolar hydrolase activities) under nutrient stress conditions. The functional redundancy between Vps21 and Ypt53 is also underscored by the fact that overexpressed Ypt53 from the VPS21 promoter rescued the defect of CPY trafficking in vps21⌬ypt52⌬ypt53⌬ triple mutant cells (Fig. 2B). In the case of another Rab5 isoform, Ypt52, the FIGURE 5. Ypt53 and Vps21p are required to maintain the respiratory activity of mitochondria during prolonged incubation of cells in nitrogen starvation medium. A, cells were grown in synthetic complete medium, transferred to SD-N ϩ 50 mM MES-KOH medium (pH 6.2), and incubated further for 2 days. Subsequently, cells were spread on a YPglucose agar plate (minus adenine), and the plates were incubated for 4ϳ5 days. Cells that contain ade2 mutations accumulate a red pigment on the YPglucose plate. However, when cells lack respiratory activity, they form white colonies. B, the percentage of white colonies was calculated. The bar graph shows mean Ϯ S.D. of three independent experiments (error bars). C, cells from red and white colonies were streaked on YPglucose and YPglycerol plates to test the respiratory defect. Representative plates are shown. Red colonies were able to grow on both YPglucose and YPglycerol plates, whereas white colonies were able to grow on the YPglucose plate but not on the YPglycerol plate, indicating that cells in white colonies are respiratory-deficient. GTPase activity is negatively regulated by Roy1, a non-Skp1-Cul1-F-box-type F-box protein, under normal conditions (19). It is currently unknown how this negative regulation is canceled. However, the deletion of ROY1 could also partially rescue the vps21⌬ phenotypes (19), implying a potential role of Ypt52 in class C core vacuole/endosome tethering. In fact, GTPlocked Ypt52 has also been shown to bind Vps8 (13). It should be noted that the levels of Ypt52 and Roy1 were unaltered under nutrient-limited conditions (data not shown). Therefore, the activation of Ypt52 might occur under conditions other than nutrient stress.
Although it is quite likely that the up-regulated Ypt53 contributes to the activation of vacuolar activity, our results also raised the possibility that Rab5 might function in the release of the autophagic body from the vacuolar membrane into the lumen. It has been proposed that, in mammals, autophagy can be activated downstream of Rab5 signaling through the beclin1-Vps34-containing complex (46,47). Further analysis is required to clarify a potential direct role of Rab5 in both yeast and mammals, but we consider that the Rab5-dependent activation of vacuolar activity and Rab5-mediated completion of autophagy can occur simultaneously and not in a mutually exclusive manner. In any event, our observations should form a basis for further investigation of a role of Rab5 in vacuolar activities.