Article |
Address correspondence to Chris A. Kaiser 68-533, Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: (617) 253-9804. Fax: (617) 253-6622. E-mail: ckaiser{at}mit.edu
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Abstract |
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Key Words: GAP1; rapamycin; GLN3; Golgi; RTG1
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Introduction |
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The nitrogen source in the growth medium also regulates the intracellular sorting of Gap1p. When cells are grown on glutamate or when Gap1p is artificially expressed during growth on glutamine, Gap1p is sorted to the vacuole and Gap1p activity at the plasma membrane is very low. Conversely, during growth on the nitrogen sources urea, proline, or ammonia (in the S288C background), Gap1p is sorted to the plasma membrane and Gap1p activity at the plasma membrane is high (Stanbrough and Magasanik, 1995; Roberg et al., 1997b; Chen and Kaiser, 2002). Gap1p sorting is thought to be largely regulated at the endosome or trans-Golgi stages of the secretory and endosomal trafficking pathways (Roberg et al., 1997b; Helliwell et al., 2001).
Mutations that alter Gap1p sorting can be broadly divided into two classes: mutations that affect Gap1p trafficking, and mutations that affect the production of the sorting signal (Magasanik and Kaiser, 2002). The first class of mutations resides mainly in genes involved in ubiquitination, such as BUL1, BUL2, RSP5, and DOA4. Apparently, polyubiquitination of Gap1p is required for its sorting to the vacuole. Thus, mutations that interfere with the polyubiquitination of Gap1p cause high Gap1p activity and increased sorting of Gap1p to the plasma membrane (Helliwell et al., 2001; Soetens et al., 2001; Springael et al., 2002).
The second class of mutations that affect Gap1p sorting resides in genes that influence the net amount of amino acid biosynthesis, such as GDH1, GLN1, and MKS1 (Chen and Kaiser, 2002). Yeast uses glutamate and glutamine as the nitrogen donors to synthesize all other amino acids (Magasanik, 1992). Thus, mutations that affect the rate of glutamate and glutamine synthesis also affect the net synthesis of all amino acids, and therefore affect Gap1p sorting. GDH1 encodes the anabolic glutamate dehydrogenase, the primary enzyme responsible for glutamate synthesis during growth on ammonia medium (Grenson et al., 1974). GLN1 encodes glutamine synthetase, an essential gene for growth on medium lacking glutamine (Mitchell, 1985). Deletion of GDH1 or mutation of GLN1 causes a decrease in cellular glutamate and/or glutamine content and an increase in sorting of Gap1p to the plasma membrane relative to wild type. Mutation of MKS1 has the opposite effect. MKS1 encodes a negative regulator of the Rtg1/3p transcription factors that control the expression of the TCA cycle enzymes responsible for -ketoglutarate synthesis during growth on medium with glucose (Liu and Butow, 1999; Dilova et al., 2002; Sekito et al., 2002; Tate et al., 2002). Because
-ketoglutarate forms the carbon skeleton from which glutamate and glutamine are derived, MKS1 has a net negative effect on glutamate and glutamine synthesis. Thus, an mks1
mutant shows high intracellular amino acid levels and decreased sorting of Gap1p to the plasma membrane (Chen and Kaiser, 2002).
One of the first mutants with a defect in Gap1p trafficking that we isolated was the lst8-1 mutant. The lst8-1 mutant was shown to greatly diminish sorting of Gap1p to the plasma membrane in cells grown on ammonia or urea as a nitrogen source (Roberg et al., 1997a). LST8 encodes an essential protein with WD-repeats and has a closely related human orthologue. A recent report from the Butow lab showed that Lst8p was involved in the negative regulation of the Rtg1/3p transcription factors (Liu et al., 2001). The lst8 mutants they isolated were shown to have elevated CIT2 transcription and decreased sensitivity to glutamate for CIT2 repression (Liu et al., 2001). These findings suggested that either Lst8p has an indirect effect on Gap1p sorting, like Mks1p, or that Lst8p has at least two separate functions: one function in the regulation of Rtg1/3p, and another function in the regulation of permease sorting.
Recently, Hall and colleagues reported that Lst8p was associated with Tor1p and with Tor2p. Tor1p coimmunoprecipitated with Kog1p and Lst8p, and Tor2p coimmunoprecipitated with Kog1p, Lst8p, Avo1p, Avo2p, and Avo3p. These coimmunoprecipitation studies, supplemented by depletion assays with genes under the control of glucose-repressible promoters, led Hall and colleagues to propose that Lst8p associates with the Tor proteins in the TORC1 and TORC2 complexes, which may have distinct roles in growth control (Loewith et al., 2002).
Here, we investigate the role of Lst8p in Gap1p permease sorting and find that the effects of lst8 mutations on Gap1p sorting are an indirect consequence of increased intracellular amino acid levels in lst8 mutants. We characterize the phenotypes of lst8 mutants and present evidence that Lst8p is a positively acting component of Tor-containing complexes.
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Results |
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Identification of temperature-sensitive lst8 mutations
LST8 is an essential gene, and we generated conditional alleles to investigate the essential function of LST8. The lst8-6 and lst8-7 temperature-sensitive alleles were generated by hydroxylamine and PCR mutagenesis, respectively, and integrated at the LST8 locus. The lst8-6 mutant failed to grow at temperatures above 34°C, whereas lst8-7 failed to grow above 37°C on YPD medium. Like lst8-1, both lst8-6 and lst8-7 had low Gap1p activity at the plasma membrane during growth on ammonia medium at 24°C (unpublished data).
We first checked lst8-6 and lst8-7 for derepressed Rtg1/3p activity by using a PCIT2-LacZ reporter. Rtg1/3p activity is normally repressed in the presence of glutamate, but has been reported to be derepressed in several different lst8 mutants grown in YPD or in medium with casamino acids (Liu et al., 2001). We found that lst8-6 and lst8-7 had strongly derepressed PCIT2-LacZ expression during growth on glutamate medium (Table II). Consistent with previous reports, we found that the lst8-1 allele showed little Rtg1/3p derepression, and that overall the lst8 mutants had a more modest effect on Rtg1/3p derepression on glutamate medium than mks1 (Liu et al., 2001; Sekito et al., 2002). The lst8-6 and lst8-7 mutants were able to suppress rtg2
somewhat with regard to PCIT2-LacZ activity, though suppression was not complete as in the case of mks1
(Table II; Liu et al., 2001; Sekito et al., 2002). Also, lst8-6 and lst8-7 (but not lst8-1) complemented the glutamate auxotrophy of rtg2
(unpublished data).
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We found that inclusion of sorbitol in the growth medium fully rescued the temperature sensitivity of lst8-6 and lst8-7, indicating that these mutations were lethal at high temperature due to cell lysis (Fig. 5 A). However, inclusion of sorbitol in the growth medium did not restore growth to an lst8 mutant (unpublished data), indicating that the lst8
mutant fails to grow for reasons other than cell wall instability. We found that SDS in the growth medium or fks1
or cwh41
mutations could partially suppress the temperature sensitivity of lst8-6 (Fig. 5, B and C). Together, these data suggest that Lst8p may also participate with Tor2p in maintaining cell wall integrity.
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To test this prediction, we measured Gap1p activity in a strain containing PADH1-HA-GAP1 grown for 18 h in ammonia medium with a sublethal concentration of 5 ng/ml rapamycin. At this low rapamycin concentration, the doubling time is 90% of the doubling time seen in ammonia medium without rapamycin (unpublished data). Cells grown in the sublethal concentration of rapamycin showed 6% of the Gap1p activity of cells without rapamycin (Fig. 7 A), and a corresponding decrease in the amount of Gap1p localized to the plasma membrane (Fig. 7 B). As with lst8-1, the Gap1p sorting defect caused by the low level of rapamycin could be partially suppressed by gdh1
(Fig. 7 A). Furthermore, cells grown in ammonia medium plus 5 ng/ml rapamycin for 18 h showed a 2.6-fold increase in total amino acid content relative to cells grown without rapamycin (Table I). Thus, like mutation of lst8, impairment of the TOR pathway by growth with a sublethal rapamycin concentration causes increased amino acid levels and decreased Gap1p sorting to the plasma membrane.
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To test whether other lst8 alleles show genetically distinct interactions with the Rtg1/3p and Gln3p pathways, we isolated additional lst8 temperature-sensitive alleles by PCR mutagenesis and performed PCIT2-LacZ reporter assays in glutamate and PGAP1-LacZ reporter assays in glutamine. All mutants were sequenced and represent independent alleles with 14 missense mutations each (unpublished data). We found that, like lst8-6, the lst8-8, lst8-9, lst8-11, lst8-13, and lst8-16 mutants showed strong defects in Rtg1/3p regulation, but only modest defects in Gln3p regulation (Table V). In contrast, the lst8-15 mutant showed a modest defect in Rtg1/3p regulation, but a stronger defect in Gln3p regulation. Thus, lst8 alleles appear to differentially affect the Rtg1/3p and Gln3p transcription pathways, suggesting that Lst8p may be the component that transduces the different outputs of the Tor1/2p complex.
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We also used fluorescence microscopy to examine Lst8p localization. A GFPLst8p fusion, which complements an lst8 mutation, was localized to discrete bodies, some adjacent to the vacuole (Fig. 8 D), consistent with the cofractionation of Lst8p with Golgi and endosomal membranes.
We performed flotation gradients with a strain coexpressing HA-Lst8p and HA-Tor1p, and found that HA-Lst8p and HA-Tor1p cofractionate with each other (Fig. 8 E). The peak of Tor1p in fractions 36 is broader than the Lst8p peak, perhaps indicating that Tor1p is also present on other membranes that lack Lst8p or that more Tor1p dissociated from membranes while floating up through the gradient. Like Lst8p, Tor1p has significant overlap with the endosomal, Golgi, and vacuolar marker proteins Pep12p, GDPase, and Vph1p (Fig. 8 E). We also tested whether the lst8-1, lst8-6, and lst8-7 mutations changed the fractionation pattern of Tor1p in these flotation gradients, but found no significant effect (unpublished data). Thus, Lst8p is a membrane-associated protein that appears to localize to the Golgi or endosomal compartments with Tor1p.
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Discussion |
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How do Lst8p and Tor1/2p act together to regulate Rtg1/3p and Gln3p activity? Our analysis of a collection of lst8 mutants showed that some alleles had a greater effect on Rtg1/3p-dependent transcription than on Gln3p-dependent transcription, whereas other alleles had a greater effect on Gln3p-dependent transcription (Table V). The qualitatively different interactions of Lst8p with these two regulatory pathways imply that Lst8p has two genetically separable functions; apparently different parts of the Lst8 protein interact with either the Rtg1/3p or Gln3p regulatory pathways. This finding suggests that Lst8p is the subunit of the TOR complex that communicates with the downstream effectors of the TOR pathway.
The TOR proteins are thought to have two distinct functions. One function, the integration of nutrient signals with cell growth, can be performed by either Tor1p or Tor2p and is inhibited by rapamycin treatment. The second function, the maintenance of the actin cytoskeleton and cell wall integrity, is unique to Tor2p and is not inhibited by rapamycin (Zheng et al., 1995; Schmelzle and Hall, 2000). Our finding that lst8 mutants exhibit both the properties of rapamycin-treated cells and the defects in cell wall integrity of tor2 mutants, and our finding that Lst8p associates with both Tor1p and Tor2p imply that Lst8p acts with the TOR gene products to promote both the shared and the Tor2p-unique function. This result is in agreement with the recent report that Lst8p is found in two types of TOR complexes, proposed to fulfill the two different TOR functions (Loewith et al., 2002). However, although Loewith et al. (2002) observed depolarized actin in cells with PGAL1-LST8 after 15 h in glucose, we did not see any actin defects in lst8 mutants at 24°C in ammonia medium (unpublished data), the same conditions under which we see a strong Gap1p sorting defect. These results suggest that although complete depletion of Lst8p may eventually cause actin depolarization, the Gap1p sorting defect we observe in lst8 mutants is not due to a cytoskeletal defect.
Rtg2p is a positive regulator of Rtg1/3p activity, but there is conflicting experimental data on the relationship between TorLst8p and Rtg2p in the regulation of Rtg1/3p. Powers and colleagues found that inactivation of the TOR pathway by rapamycin in an rtg2 mutant fails to induce CIT2 expression by Rtg1/3p, and concluded that rtg2
is epistatic to TOR inactivation (Komeili et al., 2000; Dilova et al., 2002). On the other hand, Liu et al. (2001) found that mutation of lst8 could restore CIT2 expression to an rtg2
mutant, and therefore concluded that lst8 is epistatic to rtg2
. Like Liu et al., we found that some lst8 alleles could restore CIT2 expression in an rtg2
background (Table II), which again suggested that lst8 is epistatic to rtg2
. One possible explanation for the conflicting epistasis results with rtg2
is that abrupt inactivation of TOR by treatment with rapamycin may have a different effect on the regulatory network controlling Rtg1/3p activity than constitutive inactivation of TOR complex activity by an lst8 mutation. We considered the possibility that Rtg2p might be a general negative regulator of TorLst8p, and tested for effects of rtg2
mutants on Gln3p-dependent transcription, a second output of the TorLst8p pathway. However, we did not observe an effect of a rtg2
mutation on Gln3p activity using a PGAP1-LacZ reporter on glutamate, indicating that Rtg2p specifically regulates Rtg1/3p. The existing data regarding Rtg2p is compatible with a model in which Rtg2p acts as a negative regulator of Mks1p (Dilova et al., 2002; Sekito et al., 2002), and Rtg2p and Mks1p act in parallel to TorLst8p to regulate Rtg1/3p activity (Fig. 9). Clarification of the precise relationship between Rtg2p and the TOR pathway awaits further biochemical characterization of Rtg2p.
We found that Lst8p is associated with membranes and appears to localize to the endosomal/Golgi compartments. Tor1p also cofractionates with Lst8p and with endosomal/Golgi and vacuolar markers (Fig. 8). Previous studies of Tor1/2p localization have led to a variety of conclusions about the identity of the membranes with which Tor is associated: Cardenas and Heitman (1995) reported that Tor2p associates with vacuolar membranes and Kunz et al. (2000) reported that Tor1p and Tor2p associate with the plasma membrane and with a second, unidentified membrane compartment. Recently, Loewith et al. (2002) reported that a pool of Lst8p eluted separately from the Tor proteins during gel filtration of a lysate prepared by agitation with glass beads, suggesting that not all the Lst8p is associated with Tor1/2p or that some Lst8p dissociated from Tor1/2p during lysate preparation. Kog1p/mRaptor is also a TOR-associated protein with WD-repeats whose association with TOR is sensitive to nutrient conditions (Hara et al., 2002; Kim et al., 2002). Thus, Lst8p may act with Tor1/2p and Kog1p/Raptor as a component of a large complex on endosomal/Golgi membranes for sensing intracellular nutrients and signaling to metabolic pathways.
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Materials and methods |
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Assays for amino acid uptake, ß-galactosidase, and total amino acid content
Strains were cultured to 48 x 106 cells/ml, washed twice with nitrogen-free medium by filtration on a 0.45-µm nitrocellulose filter (Millipore), and amino acid uptake assays were performed as described previously (Roberg et al., 1997b). ß-Galactosidase activity was measured with the permeabilized cell method (Adams et al., 1996). Two independent transformants were grown at RT (22°C) and assayed in duplicate. Each experiment was performed 25 times with similar results. Total amino acid analysis was performed as described previously (Chen and Kaiser, 2002).
Immunofluorescence and fluorescence microscopy
Immunofluorescence was performed using standard protocols (Adams et al., 1996) with the following modifications. PBS + 2% BSA was used for blocking and for diluting antibodies, cells were incubated with primary antibody overnight at 4°C, and samples were washed 15 times after each antibody incubation. Antibodies used were purified monoclonal 9E10 (Zymed Laboratories) and Alexa® 488conjugated goat antimouse IgG (Molecular Probes, Inc.). For GFP microscopy, cells were grown in SMM-leucine media overnight to exponential phase, then Tris-HCl, pH 8.0 was added to 100 mM and NaN3 was added to 1% for 15 min before viewing to ensure that GFP was folded and to enhance detection of GFP in acidic compartments (Bilodeau et al., 2002). Images were collected using a fluorescence microscope (Eclipse E800; Nikon), a digital camera (Hamamatsu Corporation), and Openlab software (Improvision).
Equilibrium density centrifugation, differential centrifugation, and extraction of proteins from the particulate fraction
Protocols are described in Kaiser et al. (2002). For the differential centrifugation and the extraction of proteins from the particulate fraction protocols, cells were lysed by spheroplasting and douncing. Antibodies used were: mouse anti-HA 16B12 (Covance); mouse anti-HA 12CA5 (BAbCo); rabbit anti-Pma1p (a gift of S. Losko and R. Kölling, Heinrich-Heine-Universitat, Düsseldorf, Germany), mouse anti-Dpm1p (Molecular Probes, Inc.); mouse anti-Pgk1p (Molecular Probes, Inc.); mouse anti-Vph1p (Molecular Probes, Inc.); and rabbit anti-Pep12p. Anti-Pep12p serum was made using a standard antibody protocol (Covance) with 6xHis-Pep12p made from truncated PEP12 in pET24a (a gift of M. Lewis and H. Pelham, MRC Laboratory of Molecular Biology, Cambridge, UK).
Flotation gradient with Lst8p or Tor1p-containing membranes
Flotation gradients were performed as described previously (Kaiser et al., 2002), with the following modifications: 3 x 109 cells from a logarithmically growing culture were harvested by filtration, then washed in ice-cold de-energizing buffer (50 mM Tris-HCl, pH 7.5, 10 mM NaN3, and 10 mM KF), and washed in ice-cold STE10 (10% wt/wt sucrose, 10 mM Tris-HCl, pH 7.5, and 10 mM EDTA, pH 8.0) by centrifugation. Cells were lysed by agitation with glass beads in 0.75 ml lysis buffer (STE10 with PMSF and pepstatin). 2.25 ml lysis buffer was added, and the lysate was cleared by centrifugation at 500 g for 3 min. Membranes were collected by layering 2 ml of the cleared lysate onto a cushion of 0.2 ml STE80 (80% wt/vol sucrose, 10 mM Tris-HCl, pH 7.5, and 10 mM EDTA) and centrifuging in a TLS-55 rotor (Beckman Coulter) at 100,000 g for 1 h at 4°C. Membranes that collected at the interface were combined with enough STE80 to make the density of the solution equivalent to the density of STE50 (50% wt/wt sucrose, 10 mM Tris-HCl pH 7.5, and 10 mM EDTA). A volume of membrane solution corresponding to 3 x 108 cells was loaded at the bottom of a 3050% (wt/wt) continuous sucrose gradient and centrifuged at 100,000 g for 17 h with no brake in an SW55Ti rotor (Beckman Coulter). Fractions were collected manually from the top of the gradient. A portion of each fraction was subject to TCA precipitation, SDS-PAGE, and Western blotting, or was assayed for GDPase and Kex2p activity as described previously (Kaiser et al., 2002). Intensity of protein bands on Western blots was quantitated using the Kodak Image Station 440 imaging system and Kodak 1D software (PerkinElmer).
Immunoprecipitation and immunoblotting of Lst8-associated proteins
Cells (108) growing logarithmically in SMM-uracil medium were harvested and washed with ice-cold 50 mM Hepes, pH 7.5, and 10 mM NaN3. Cells were lysed by agitation with glass beads in Co-IP buffer (20 mM Hepes, pH 6.8, 80 mM potassium acetate, 5 mM magnesium acetate, and 0.5% CHAPS) with protease inhibitors (1 mM PMSF, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 2 µg/ml aprotinin), then the lysate was diluted to 1 ml with Co-IP buffer with protease inhibitors. The lysate was cleared by centrifugation at 13,000 g for 3 min, then the supernatant was precleared by incubation with protein A Sepharose for 30 min at 4°C. A portion of the precleared lysate was removed as the "total" sample. To the remaining lysate, rabbit anti-myc antibody (9E10, Santa Cruz Biotechnology) was added and incubated for 2 h at 4°C. Then, Protein A Sepharose was added and the mixture incubated for 1 h at 4°C. Immunoprecipitates were washed three times with Co-IP buffer + 0.1% CHAPS, and once with detergent-free Co-IP buffer. Immunoprecipitates were solubilized by incubation in sample buffer for 30 min at 37°C and resolved by SDS-PAGE. Antibodies used for immunoblotting were mouse anti-HA 12CA5 and mouse anti-myc 9E10 (Covance).
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Acknowledgments |
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This work was supported by a Howard Hughes Medical Institute predoctoral fellowship (to E.J. Chen) and by a National Institutes of Health grant GM56933 (to C.A. Kaiser).
Note added in proof. In a recent report, T. Powers and colleagues (Wedaman, K.P., A. Reinke, S. Anderson, J. Yates 3rd, J.M. McCaffery, and T. Powers. 2003. Mol. Biol. Cell. 14:12041220) used immunogold electron microscopy to colocalize Lst8p and Tor2p to punctate, membranous sites adjacent to (but distinct from) the plasma membrane and other sites within the cell. This localization of Tor/Lst8 is consistent with the endosomal/Golgi localization for Lst8p based on membrane fractionation we report in this paper.
Submitted: 25 October 2002
Revised: 24 February 2003
Accepted: 24 February 2003
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