Article |
Address correspondence to Akihiko Nakano, Molecular Membrane Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-9547. Fax: 81-48-462-4679. E-mail: nakano{at}postman.riken.go.jp
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Abstract |
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Key Words: ERG6; Tat2p; raft; multivesicular body; ubiquitin
K. Umebayashi's present address is Department of Cell Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
* Abbreviations used in this paper: MVB, multivesicular body; MVL, mevalonic acid lactone; VPS, vacuolar protein sorting.
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Introduction |
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For gaining further insights into the sterol-dependent sorting processes, the yeast Saccharomyces cerevisiae is an attractive organism. The structure of the major sterol in yeast, ergosterol, is slightly different from cholesterol, but its biosynthetic pathway has been almost completely understood (for review see Daum et al., 1998). In terms of membrane trafficking, the sterol composition has been shown to affect endocytosis in yeast (Heese-Peck et al., 2002). Evidence is also presented that yeast does have lipid rafts that are important for protein sorting (Bagnat et al., 2000, 2001). To further understand the role of sterols in traffic, we decided to start a study paying attention to yeast erg mutants, which are defective in the ergosterol biosynthesis. We examined phenotypes of the erg mutants to find potential defects in protein sorting. We were aware that the erg6 mutant was known to show reduced uptake of tryptophan from the medium (Gaber et al., 1989). The ERG6 gene encodes S-adenosylmethionine 24 methyltransferase, which acts at a late step of the ergosterol biosynthetic pathway by converting zymosterol to fecosterol. The tryptophan uptake defect raised the possibility that the high affinity tryptophan permease Tat2p (Schmidt et al., 1994) is not correctly targeted to the plasma membrane. In this article, we will show the results of our detailed analysis of the localization of Tat2p and its post-Golgi sorting, with a particular focus on the roles of ubiquitination and lipid raft association.
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Results |
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When wild-type cells were shifted from high to low tryptophan medium, staining of cell periphery became evident (Fig. 3 D), indicating that Tat23HAp is now targeted to the plasma membrane. This is reasonable because under the low tryptophan condition, Tat23HAp should be on the plasma membrane for efficient uptake of tryptophan.
These results demonstrate that the plasma membrane localization of Tat23HAp is regulated by the tryptophan concentration in the medium. Tat23HAp is targeted to late endosomes at high tryptophan, and to the plasma membrane at low tryptophan.
Tat2p is missorted to the vacuole in the erg6 mutant
Next, we examined the localization of Tat23HAp in erg6 cells (Fig. 4 A). At high tryptophan, Tat23HAp was localized to punctate structures as in wild-type cells. However, when the
erg6 cells were shifted to the low tryptophan medium, the staining of Tat23HAp did not change to the plasma membrane pattern, and the fluorescence within the cells became very faint.
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In contrast, in erg6 cells, the amount of Tat23HAp was markedly reduced at low tryptophan (Fig. 4 B), consistent with the faint signal in the immunostaining. This reduction is due to vacuolar degradation because the disruption of PEP4 in
erg6 prevented the loss of Tat23HAp at low tryptophan. Thus, in
erg6 cells, Tat23HAp is missorted to the vacuole and quickly degraded under the low tryptophan condition.
The missorting of Tat2p implies that the severe tryptophan auxotrophy of erg6 can be suppressed if the vacuolar delivery is blocked. By using the pep12 mutation that inhibits the traffic to late endosomes and thereby redirects vacuolar proteins to the cell surface (Becherer et al., 1996), we show this is indeed the case. As shown in Fig. 4 C, the
erg6 mutant did not grow below 100 µg/ml of tryptophan. However, this defect was clearly suppressed by
pep12, although not completely. The
erg6
pep12 double mutant grew well at 100 µg/ml and slowly at 50 µg/ml.
Sorting of Tat2-GFP late in the secretory pathway
To follow the route of Tat2p in more detail, the localization of another fusion construct, Tat2-GFP, was examined in various mutants defective in late steps of the secretory pathway. The results are shown in Fig. 5. In wild-type cells grown at high tryptophan, Tat2-GFP was localized to the vacuole as well as to perivacuolar late endosomes. When the cells were shifted to low tryptophan, Tat2-GFP was localized to the plasma membrane. The advantage using Tat2-GFP in living cells is that the plasma membrane was much more clearly visualized than in the fixed cells by immunostaining (compare with Fig. 3 D). This is probably because the enzymatic removal of the cell wall can be omitted if Tat2-GFP is used. Again, relocalization of Tat2-GFP to the plasma membrane in response to low tryptophan was not observed in erg6 cells, with prominent fluorescence in the vacuole.
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To test whether Tat2-GFP is targeted to the plasma membrane by the exocytic pathway, temperature-sensitive sec mutants were examined. SEC14 is required for the exit from the Golgi (Stevens et al., 1982). When the sec14 mutant was shifted to the low tryptophan medium and grown at the permissive temperature 23°C, plasma membrane localization of Tat2-GFP was observed. However, when the low tryptophan medium was kept at the nonpermissive temperature 37°C, Tat2-GFP was not targeted to the plasma membrane and stayed in intracellular compartments. SEC6 encodes a component of the "exocyst" (TerBush et al., 1996), which is required for the fusion of Golgi-derived vesicles with the plasma membrane. At 37°C in the low tryptophan medium, Tat2-GFP was not targeted to the plasma membrane of the sec6 mutant. These results appear to indicate that Tat2-GFP follows the exocytic pathway to the plasma membrane at low tryptophan.
In pep12 cells, Tat2-GFP was localized to the plasma membrane irrespective of the tryptophan concentration. Thus, the inhibition of the vacuolar delivery by
pep12 resulted in constitutive plasma membrane targeting of Tat2-GFP. Consistent with the suppression of
erg6 by
pep12 (Fig. 4 C), Tat2-GFP was also targeted to the plasma membrane in
erg6
pep12 cells.
The vacuolar protein sorting (VPS)* pathway represents the direct vesicular traffic from the trans-Golgi to late endosomes. The VPS1 gene product is involved in this pathway and is considered to be necessary for the vesicle formation from the trans-Golgi (Nothwehr et al., 1995). Unlike in the pep12 mutant, Tat2-GFP was not missorted to the plasma membrane in
vps1 cells at high tryptophan. Tat2-GFP was seen in the vacuole as well as perivacuolar dots. When the
vps1 cells were shifted to low tryptophan, plasma membrane localization of Tat2-GFP was observed.
vps1 did not suppress the severe tryptophan auxotrophy of
erg6, either (unpublished data).
The result with vps1 indicates that Tat2-GFP does not follow the normal VPS pathway to reach late endosomes at high tryptophan. The
pep12 mutant is defective not only in the VPS pathway, but also in the endocytic pathway. Traffic from early to late endosomes is blocked in
pep12 (Gerrard et al., 2000). Thus, Tat2p must have taken the route from the trans-Golgi to late endosomes at high tryptophan via early endosomes. We would suggest that the tryptophan-dependent sorting of Tat2p occurs in early endosomes, and the
erg6 mutant is defective in this sorting process (see Discussion and Fig. 9).
Ubiquitination and sorting of Tat2p
Evidence is rapidly accumulating that ubiquitin acts as a sorting signal at multiple steps in post-Golgi traffic. In the case of the yeast general amino acid permease Gap1p, sorting is affected by its ubiquitinated status (Helliwell et al., 2001; Soetens et al., 2001). Polyubiquitination of Gap1p by the Rsp5p ubiquitin ligase complex results in sorting to the vacuole instead of the plasma membrane. In other words, polyubiquitin is recognized as a vacuolar-targeting signal. This prompted us to examine the ubiquitination status of Tat2p. Tat23HAp was immunoprecipitated from cells expressing myc-tagged ubiquitin (Hochstrasser et al., 1991), and the precipitated materials were detected with the anti-myc or anti-HA antibody. To prevent degradation of Tat23HAp in the vacuole, pep4 strains were used. As shown in the right panel of Fig. 6 A, Tat23HAp was specifically precipitated (compare lane 1 with lanes 24). High mol wt mycubiquitin conjugates of Tat23HAp were detected in
pep4 cells grown under the high tryptophan condition, (Fig. 6 A, left panel, lane 3), indicating that Tat23HAp is polyubiquitinated. It is known that ubiquitination of cargo proteins, such as the yeast pheromone receptor Ste2p, occurs in the plasma membrane on endocytic internalization (Hicke and Riezman, 1996). However, because Tat23HAp does not take the detour to the plasma membrane by exocytosis and endocytosis under the high tryptophan condition (Fig. 3), the place of its polyubiquitination must be somewhere else. On the other hand, the polyubiquitination of Tat23HAp was not detected in sec14 (Fig. 6 A, lane 5), indicating that the polyubiquitination reaction takes place after Tat23HAp has left the Golgi.
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Next, we examined the effect of bul1 on the tryptophan auxotrophy of
erg6. Surprisingly, the
erg6
bul1 double mutant could grow at as low as 10 µg/ml tryptophan (Fig. 6 C). Similarly, the deletion of DOA4, which reduces the efficiency of overall protein ubiquitination, also suppressed the severe tryptophan auxotrophy of
erg6, although weakly.
erg6
doa4 cells could grow at 20 µg/ml of tryptophan (Fig. 6 C). These results led us to the hypothesis that Tat2p is inappropriately polyubiquitinated in
erg6, resulting in the missorting to the vacuole.
There is another line of evidence that indicates aberrant polyubiquitination of Tat2p in erg6. Many lysine residues are present in the cytoplasmic domains of Tat2p, among which Beck et al. (1999) identified five lysine residues (10, 17, 20, 29, and 31) in the NH2-terminal domain as the ubiquitin acceptor sites on nutrient starvation. We confirmed that the three lysine residues (10, 17, and 20) are indeed the major ubiquitin acceptor sites of Tat2p. The variant of Tat23HAp, in which these three lysine residues were replaced by arginine (Tat23K>R-3HAp), was little ubiquitinated in the
pep4 background (Fig. 6 D, compare lane 2 and lane 3). However, in
erg6
pep4 cells, Tat23K>R-3HAp was again clearly ubiquitinated (Fig. 6 D, lane 4). The ubiquitination of Tat2p in
erg6 must have occurred on improper lysine residues.
Missorting to the multivesicular body sorting pathway in the erg6 mutant
In immunofluorescence staining of pep4 strains to visualize vacuolar localization of Tat23HAp, we noticed an interesting difference between ERG6 and
erg6 cells (Fig. 7 A). In
pep4 cells, the vacuolar-limiting membrane was clearly stained, regardless of the tryptophan concentration. In contrast, in the
erg6
pep4 cells, the fluorescence of Tat23HAp was not detected on the vacuole-limiting membrane, but almost exclusively in the lumen, either at high or low tryptophan. Such lumenal staining would indicate that Tat23HAp entered the multivesicular body (MVB)sorting pathway, which transfers a subset of cargo proteins to the invaginating vesicles in yeast late endosomes (Odorizzi et al., 1998). To test this possibility, we examined the effect of VPS27 disruption. VPS27 is one of the class E VPS genes, all of which are required for MVB formation (Odorizzi et al., 1998). As shown in Fig. 7 A, Tat23HAp was localized to the vacuole-limiting membrane and the exaggerated class E compartment in
erg6
vps27
pep4. These results indicate that Tat23HAp is efficiently sorted to the MVB pathway in
erg6, regardless of the tryptophan concentration.
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In contrast to Tat23HAp, it may be noted that the fluorescence of Tat2-GFP is clearly seen in the vacuole lumen of wild-type cells (Fig. 5). We raised a specific antibody against Tat2p, and found that untagged Tat2p was detected on the vacuolar-limiting membrane in pep4, but in the vacuole lumen in
erg6
pep4 (Fig. 7 C). This behavior is very similar to that of Tat23HAp, and therefore, the results with Tat23HAp may reflect the authentic nature of Tat2p. The sorting of Tat2-GFP into the vacuolar lumen was blocked in both
vps27 and
doa4 cells (Fig. 7 D), indicating that it undergoes ubiquitin-dependent MVB sorting. Like in
bul1 cells (Fig. 6 B), the plasma membrane signal of Tat2-GFP in
doa4 cells was obvious at high tryptophan, and became remarkable on the shift to low tryptophan.
Then why is Tat2-GFP efficiently sorted to the MVB in wild-type cells, even though it is functional and correctly targeted to the cell surface at low tryptophan? Tat2-GFP may be ubiquitinated more efficiently. Alternatively, vacuolar lumenal localization of the MVB vesicles could be detected more clearly by GFP fluorescence in living cells. Due to the fixation and subsequent permeabilization procedures, the MVB vesicles might look more obscure by immunofluorescence microscopy.
As another way to assess the MVB missorting in the erg6 mutant, we looked at a different GFP marker, GFP-Pep12p. The results are shown in Fig. 7 E. As reported previously (Reggiori et al., 2000), GFP-Pep12p resides mostly on the vacuolar-limiting membrane in wild-type cells when overexpressed. However, in
erg6 cells, the GFP fluorescence was now evident in the vacuole lumen. The fluorescence on the limiting membrane still remained, indicating that GFP-Pep12p is not completely relocated to the vacuole lumen. The lumenal signal in
erg6 cells disappeared by either
vps27 or
doa4. Thus, GFP-Pep12p in
erg6 cells is also missorted to the MVB in a ubiquitin-dependent manner, indicating that a subset of cargo proteins is inappropriately ubiquitinated and then sorted to the MVB in
erg6.
Transport of Tat2p to the plasma membrane depends on detergent-insoluble membrane domains
Because the deficiency of normal sterol in erg6 affected the sorting of Tat2p, we further investigated whether sterol-rich, detergent-insoluble membrane domains (so-called rafts) are involved in the plasma membrane delivery of Tat2p. Detergent insolubility of Tat2p was examined by treatment with CHAPS followed by a flotation analysis as diagrammed in Fig. 8 A. In wild-type cells (Fig. 8 B), a fraction of the GPI-anchored protein Gas1p floated to the interphase between 0 and 30% of OptiPrepTM (Fig. 8 B, fraction 2, arrowhead), whereas the vacuolar alkaline phosphatase Pho8p did not, as reported previously (Bagnat et al., 2000). Tat23HAp did not float to fraction 2 under the high tryptophan condition where it is sorted to late endosomes. However, Tat23HAp was clearly detected in fraction 2 under the low tryptophan condition where it is targeted to the plasma membrane. In the
erg6 mutant (Fig. 8 C), Gas1p was still detected in the floating fraction 2. This is consistent with a recent report (Sievi et al., 2001), and indicates that the sterol intermediates accumulating in the
erg6 mutant can replace ergosterol in the context of detergent-insoluble membrane domain formation. However, Tat23HAp in
erg6 cells failed to float to fraction 2 even at low tryptophan (Fig. 8 C).
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To confirm the role of rafts, we also attempted to disrupt the detergent-insoluble domain by inhibiting the initial step of the ergosterol biosynthesis. The ERG13 gene encodes HMG-CoA synthase. erg13 cells require mevalonate in the medium for growth. As reported before (Dimster-Denk et al., 1994), the growth of
erg13 cells was arrested at a low concentration (5 mg/ml) of mevalonic acid lactone (MVL). At 10 mg/ml of MVL,
erg13 cells were able to grow slowly. As shown in Fig. 8 E, only a small amount of Gas1p was found in the floating fraction 2 when MVL was supplied at 10 mg/ml. For simplicity, the distribution of Gas1p was compared between the detergent-insoluble (I) fractions (Fig. 8 A, defined as the mixture of fractions 2 and 3) and the detergent-soluble (S) fractions (Fig. 8 A, the mixture of fractions 79). As shown in Fig. 8 F, Gas1p gradually disappeared from the I fractions of
erg13 cells according to the decrease of the supplementing MVL, indicating that the detergent-insoluble domains were significantly depleted when the flux of sterol synthesis was reduced. Exactly under the same condition, Tat2-GFP was inefficiently routed to the plasma membrane, and the vacuolar staining remained prominent (Fig. 8 G). In addition,
erg13 cells were unable to grow at low tryptophan (Fig. 8 H), implying that the plasma membrane targeting of Tat2p is critical for growth under this condition. Strikingly,
bul1 suppressed such severe tryptophan auxotrophy of the
erg13 mutant (Fig. 8 H), indicating that the raft requirement for the plasma membrane targeting of Tat2p can be bypassed by the inhibition of polyubiquitination.
All these results strongly suggest that the partitioning of Tat2p into the rafts at low tryptophan is not the indirect consequence of the plasma membrane targeting, but is rather the cause of the sorting into the plasma membrane route. In other words, lipid raft sorting is very important for the cell surface delivery of Tat2p. However, it should be remembered that Tat2p can also be delivered to the plasma membrane in the absence of raft association under some conditions (for example, when the vacuolar trafficking pathway is blocked or the polyubiquitination is inhibited).
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Discussion |
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Ubiquitin-dependent sorting of amino acid permeases
It has been known that intracellular sorting of yeast nutrient transporters, such as Gap1p and the ferrichrome transporter Arn1p, is regulated late in the secretory pathway (Roberg et al., 1997; Kim et al., 2002). In the case of Gap1p, the sorting depends on the nitrogen source in the medium. Gap1p is targeted to the plasma membrane when cells are grown on urea, but to the vacuole in the glutamate medium. The similarity between Tat2p and Gap1p regarding nutrient-dependent regulation of sorting led us to suspect the presence of a common mechanism (i.e., ubiquitination).
Kaiser's group has shown that polyubiquitination by the Rsp5p ubiquitin ligase complex directs Gap1p to the vacuole instead of the plasma membrane (Helliwell et al., 2001). This also turns out to be the case with Tat2p. Bul1p, known as a component of the Rsp5p complex required for elongation of polyubiquitin chains (Yashiroda et al., 1996; Helliwell et al., 2001), plays a critical role in the regulation of ubiquitination status of Tat2p. Polyubiquitination of Tat2p is little detected in the bul1 mutant. Surprisingly, almost all the defects of
erg6 in Tat2p sorting are simultaneously suppressed by the knockout of BUL1. Presumably, aberrant polyubiquitination in the
erg6 mutant is alleviated by the
bul1 mutation. The anomaly of ubiquitination in
erg6 is also seen on the acceptor sites of ubiquitin. Tat2p is polyubiquitinated on inappropriate lysine residues in
erg6.
MVB sorting of Tat2p
erg6 cells also show a peculiar behavior in the MVB sorting. Although Tat2p remains on the limiting membrane when it is finally targeted to the vacuole in wild-type cells, Tat2p is almost completely segregated into the lumen of the vacuole in
erg6. Similarly, Pep12p is also missorted into the MVB in
erg6 cells. This MVB mistargeting is blocked by the class E
vps27 mutation, suggesting that normal mechanisms of MVB sorting by the ESCRT complexes (Katzmann et al., 2001; Babst et al., 2002a, 2002b) are operating in this process. Interestingly, a CHO cell mutant defective in cholesterol biosynthesis also shows MVB missorting of the cation-independent mannnose 6-phosphate receptor (Miwako et al., 2001). This kind of missorting may be a general outcome caused by defects of normal sterol synthesis.
Our finding that the MVB missorting of Tat2p and Pep12p in erg6 is suppressed by either
bul1 or
doa4 indicates that it occurs in a ubiquitin-dependent manner. The sequential sorting defects of Tat2p in
erg6, namely in early and late endosomes, could be explained solely by ubiquitination. That is, Tat2p is inappropriately ubiquitinated in
erg6, delivered from early to late endosomes, and then sequestered into the MVB by being caught by the ESCRT-1 complex, a putative sorting receptor for ubiquitinated cargoes (Katzmann et al., 2001).
In several cases, monoubiquitination has been shown sufficient for the entry of cargo into the MVB. Tat2p is polyubiquitinated even under the low tryptophan condition in pep4 cells (unpublished data). Beck et al. (1999) have also shown that Tat2p is polyubiquitinated under the starvation condition and found on the vacuolar-limiting membrane. Then the question is why Tat2p is not always sorted to the MVB pathway. Mono- vs. polyubiquitination could explain the difference. Alternatively, the position of ubiquitination may be important. For example, ubiquitin signals near the membrane could be recognized by the MVB-sorting machinery more easily than the distal ones (Reggiori and Pelham, 2002). For Tat2p, the major ubiquitin acceptor sites for the wild-type are Lys10, Lys17, and Lys20, all near the NH2 terminus. Ubiquitination of Tat2p in the
erg6 mutant might occur on lysine residues proximal to the membrane, resulting in more efficient MVB sorting.
Lipid raftdependent sorting of Tat2p
We present two lines of evidence indicating that association with the detergent-insoluble membrane domain is required for the plasma membrane delivery of Tat2p. First, Tat2p became detergent-insoluble under the low tryptophan condition. Second, depletion of sterols by using the mevalonate auxotroph erg13 mutant disrupted the detergent-insoluble membrane domain and simultaneously blocked the plasma membrane targeting of Tat2p. The detergent insolubility and sterol dependence of this membrane domain fit well with the concept of the lipid raft (Simons and Ikonen, 1997). Our results with
erg13 indicate that Tat2p is missorted to the vacuole in the absence of lipid rafts. Similarly, the proton ATPase Pma1p is delivered to the plasma membrane in association with rafts, and missorted to the vacuole when rafts are disrupted (Bagnat et al., 2001). It appears that raft and nonraft domains are segregated for the plasma membrane and late endosomal delivery, respectively.
The raft association is not obligatory for the plasma membrane targeting of Tat2p if its vacuolar sorting is inhibited by pep12 or
bul1. The fact that the severe tryptophan auxotrophy of the raft-deficient
erg13 was suppressed by
bul1 indicates that raft association and polyubiquitination have counteracting effects in the sorting of Tat2p. On aberrant polyubiquitination, Tat2p is probably diverted from the raft-dependent plasma membrane targeting pathway to the nonraft pathway to late endosomes. Because ERG6 is involved in a late step of the ergosterol biosynthetic pathway, sterols are not depleted in
erg6, but intermediates such as zymosterol accumulate (Munn et al., 1999). These intermediates are still capable of forming rafts, judging from the detergent insolubility of the GPI-anchored protein in
erg6. However, Tat2p cannot be associated with such altered rafts any more and missorted to the vacuole. Tat2p may be just unable to reside stably in the rafts with the unusual sterol composition, or could be excluded from the rafts due to its inappropriate polyubiquitination.
Experiments with the sec mutants grown at low tryptophan indicated that Tat2p becomes associated with rafts in the Golgi. Tat2p associates with rafts even at high tryptophan in the sec14 mutant (unpublished data). Although the altered phospholipid composition of this mutant (McGee et al., 1994) might indirectly affect the raft organization, this observation suggests that Tat2p can gain access to rafts in the Golgi. On the other hand, polyubiquitination of Tat2p was not detected in the sec14 mutant, indicating that the polyubiquitination occurs after the exit from the Golgi. Thus, Tat2p would be first partitioned into rafts, and then be subjected to the ubiquitin-dependent sorting, presumably in early endosomes.
How polyubiquitin acts as a sorting signal to the nonraft, vacuolar trafficking pathway remains to be resolved. Lafont and Simons (2001) have shown that the ubiquitin ligases Cbl and Nedd4 are partitioned into rafts. Interestingly, the yeast Nedd4 homologue Rsp5p is partially resistant to detergent extraction (Wang et al., 2001), implying that polyubiquitination of Tat2p by the Rsp5pBul1p complex could occur in the rafts. Sorting receptors such as Hrs (Raiborg et al., 2002) may bind to polyubiquitin and divert cargo proteins to the nonraft membrane domains. Alternatively, Tat2p might dissociate from rafts independently of ubiquitin. The dissociation could change the environment around the molecule and would then trigger its polyubiquitination and sorting to late endosomes.
That the slight alteration in sterol structure or composition can dramatically change the destination of a plasma membrane protein raises a possibility that similar regulation could be used for differentiation of the cell surface, for example, during the development in higher eukaryotes. Our future work will aim at understanding how sterols might be involved in such higher order regulations and how they are linked to ubiquitin, a key player in the post-Golgi traffic.
While this manuscript was in preparation, Bagnat and Simons (2002) reported that Fus1p, a plasma membrane protein required for yeast mating, is largely excluded from rafts and mislocalized to the vacuole in the erg6 mutant. This behavior of Fus1p in
erg6 is similar to that of Tat2p, and supports the view that a subset of plasma membrane proteins are missorted in
erg6 to cause pleiotropic phenotypes. Indeed, the mating deficiency (Gaber et al., 1989) and the drug hypersensitivity (Kaur and Bachhawat, 1999) of
erg6 might all be explained by the missorting of plasma membrane proteins due to impaired raft association and inappropriate ubiquitination.
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Materials and methods |
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Plasmids and antibodies
Details of the various plasmid constructions and antibodies are described in the supplemental materials and methods section (available at http://www.jcb.org/cgi/content/full/jcb.200303088/DC1).
Fluorescence microscopy
Immunofluorescence microscopy was performed essentially as described before (Nishikawa and Nakano, 1991), except that permeabilization of fixed cells was performed by spheroplasting buffer containing 1% (wt/vol) BSA and 0.1% (vol/vol) Triton X-100 for 10 min at RT. Cells were observed and photographed using a photomicroscope (model BX-60; Olympus). Alternatively, the same microscope equipped with a confocal laser scanner unit (model CSU10; Yokogawa Electronic Corp.) was used. Images were acquired by a high resolution digital charge-coupled device camera (model C474295; Hamamatsu Photonics) and processed by IPLab software (Scanalytics).
Detection of the ubiquitinated forms of Tat23HAp
Detection of ubiquitinated Tat23HAp was performed basically according to the method used for the case of Gap1p (Helliwell et al., 2001). To enhance the detection, the myc-tagged ubiquitin was exogenously expressed. The expression of myc-Ub was under the control of the CUP1 promoter, which was inducible by addition of CuSO4 to the medium (Ellison and Hochstrasser, 1991). However, myc-Ub conjugates were detectable even when the promoter was uninduced, as was reported previously (Hochstrasser et al., 1991). In this work, cells were grown at the basal expression level of the CUP1 promoter. 5 x 108 cells were collected and treated with NaN3 and potassium flouride at the final concentration of 20 mM each. The cells were resuspended in 125 µl lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1.6% SDS, 6 M urea, 5 mM N-ethylmaleimide, and 0.02% NaN3) containing a protease inhibitor mixture (1 mM PMSF, 5 µg/ml chymostatin, leupeptin, antipain, and pepstatin A, and 2.5 µg/ml aprotinin), lysed by agitation with glass beads, and incubated at 37°C for 20 min. 875 µl IP dilution buffer (1.1% Triton X-100, 170 mM NaCl, 6 mM EDTA, 60 mM Tris-HCl, pH 7.4, 5 mM N-ethylmaleimide, and 0.02% NaN3, the protease inhibitor mixture) was added to the cell lysates, and insoluble material was removed by centrifugation. 750 µl supernatant was mixed with 40 µl protein G Sepharose 4 Fast Flow (Amersham Biosciences), and precleared by rotation at RT for 30 min. The samples were centrifuged, and 700 µl supernatant was incubated overnight with 10 µl rat anti-HA antibody (3F10; Roche Diagnostics) and 15 µl protein G Sepharose suspension, with rotation at 4°C. The beads were washed twice with IP buffer (1% Triton X-100, 0.2% SDS, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.4), twice with urea wash buffer (1% Triton X-100, 0.2% SDS, 2 M urea, 250 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.4) and once with high salt wash buffer (1% Triton X-100, 0.2% SDS, 50 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.4). The beads were suspended with SDS-PAGE sample buffer (2% SDS, 5% ß-mercaptoethanol, 10% glycerol, 50 mM Tris-HCl, pH 6.8, and 0.025% bromophenol blue) containing 6 M urea, and incubated at 37°C for 20 min. 24 µl of the sample was subjected to SDS-PAGE and immunoblotting with the anti-myc antibody (9E10) to detect myc-Ub conjugates. To detect Tat23HAp, the sample was diluted 15-fold and 20 µl was loaded. Anti-HA antibody (16B12) was used for immunoblotting.
Analysis of lipid rafts
5 x 108 cells were collected, treated with NaN3 and potassium flouride at a final concentration of 20 mM each, and resuspended in 275 µl TNE buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5 mM EDTA) containing a protease inhibitor mixture (1 mM PMSF and 5 µg/ml chymostatin, leupeptin, antipain, and pepstatin A). After adding glass beads, the suspension was vortexed for 30 s and was then chilled on ice for 30 s, repeating six times. Unbroken cells and debris were removed by centrifugation at 500 rpm for 5 min. The cleared lysate (175 µl) was mixed with equal volume of TNE containing 40 mM CHAPS (Sigma-Aldrich), and was then incubated at 4°C for 30 min. The tube was centrifuged at 5,000 rpm for 5 min, and 330 µl supernatant was mixed with 770 µl 50% OptiPrepTM (Nycomed Pharma)/TNE/20 mM CHAPS to give the final concentration of 35% OptiPrepTM. The solution was set on the bottom of a 3 PC tube (Hitachi Koki Co., Ltd.), and overlaid with 1.4 ml 30% OptiPrepTM/TNE/20 mM CHAPS and 0.5 ml TNE/20 mM CHAPS. The gradients were centrifuged at 4°C for 7.5 h using a rotor (model RPS65T; Hitachi Koki Co., Ltd.) at 35,000 rpm, and nine fractions (320 µl each) were collected from the top. Each fraction was mixed with 288 µl 110 mM Tris-HCl, pH 6.8/4.4% SDS/22% glycerol and 32 µl ß-mercaptoethanol, incubated at 37°C for 5 min, and subjected to SDS-PAGE.
Online supplemental materials
Plasmid construction, antibodies, and immunoblotting procedures are included in the online supplemental materials, available at http://www.jcb.org/cgi/content/full/jcb.200303088/DC1.
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Acknowledgments |
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This work was supported by grants from the Biodesign and Bioarchitect Research Projects of RIKEN and by a President's Special Research Grant of RIKEN. K. Umebayashi is a recipient of the Special Postdoctoral Researcher fellowship of RIKEN.
Submitted: 13 March 2003
Revised: 30 April 2003
Accepted: 1 May 2003
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References |
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