![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 8, 2779-2794, August 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Section of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, Davis, CA 95616; and Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
Submitted March 26, 2007;
Revised May 1, 2007;
Accepted May 7, 2007
Monitoring Editor: Reid Gilmore
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). There are two TOR kinases in budding yeast, Tor1p and Tor2p, that independently assemble with a distinct set of proteins to form what is termed TOR complex I (TORC1), the form of TOR that is inhibited by rapamycin (Wullschleger et al., 2006). Treating cells with rapamycin elicits widespread and characteristic changes in cellular behavior that mimic nutrient deprivation, including cessation of ribosome biogenesis and protein synthesis, induction of autophagy, cell cycle arrest at the G1/S boundary, and, ultimately, entry into stationary phase (Crespo and Hall, 2002; Rohde and Cardenas, 2004; Wullschleger et al., 2006). Rapamycin affects the expression of a wide array of nutrient-responsive genes as well as intracellular sorting of distinct amino acid permeases, results that are consistent with proposals that TORC1 conveys one or more nutrient-related signals within cells (Crespo and Hall, 2002; Rohde and Cardenas, 2004). Many events downstream from TORC1 are regulated by a phosphatase regulatory module that is composed of the type 2A phosphatases Pph21p and Pph22p, the related type 2A phosphatase Sit4p, and the two regulatory proteins Tap42p and Tip41p (Di Como and Arndt, 1996; Jiang and Broach, 1999; Jacinto et al., 2001; Düvel et al., 2003). TORC1 activity also intersects with several other nutrient-responsive kinases, including cAMP-regulated protein kinase A (PKA) and Sch9p (Jorgensen et al., 2004; Marion et al., 2004; Schmelzle et al., 2004; Zurita-Martinez and Cardenas, 2005; Chen and Powers, 2006). Precisely how TORC1 activity is integrated with these kinases and phosphatases remains poorly understood, and it is likely to be very complex.
Independently of its role in TORC1, Tor2p also assembles with an overlapping yet distinct set of proteins to form TORC2 (Loewith et al., 2002; Wedaman et al., 2003; Wullschleger et al., 2006), which plays a role in polarized cell growth and actin cytoskeletal organization (Loewith et al., 2002; Wullschleger et al., 2006). This Tor2p-unique activity involves signaling to components required for proper remodeling of actin, in the form of cortical patches, at the site of bud emergence (Schmidt et al., 1996; Schmidt et al., 1997; Helliwell et al., 1998; Crespo and Hall, 2002). These components include the Rho1p GTPase and its associated regulatory partners Rom2p and Sac7p, which function in part by signaling to protein kinase C (Pkc1p), an upstream activator of the mitogen-activated protein kinase (MAPK) Slt2p/Mpk1p (Helliwell et al., 1998; Pruyne and Bretscher, 2000; Crespo and Hall, 2002). More recently, the AGC kinase Ypk2p and two novel effectors, Slm1p and Slm2p, have been identified that also function downstream of TORC2 and that are required for proper actin polarization and cell viability (Audhya et al., 2004; Kamada et al., 2005). In addition, TORC2 has now been linked to a number of other cellular processes, including receptor-mediated endocytosis, phosphoinositide signaling, and calcineurin regulation as well as cell integrity maintenance (Schmelzle et al., 2002; deHart et al., 2003; Levin, 2005; Mulet et al., 2006; Tabuchi et al., 2006). Whether each of these different processes are linked to TORC2 via the Rho1p GTPase module and/or the newly discovered Slm proteins has not been determined. Interestingly, rapamycin treatment and/or inhibition of the Tap42p/Sit4p phosphatase system has also been shown to affect both actin polarization as well as cell integrity signaling, suggesting an unexpected degree of overlap with respect to functions carried out by TORC1 versus TORC2 (Torres et al., 2002; Wang and Jiang, 2003). In this regard, a recent chemical genetic screen has identified a large number of yeast gene deletion mutants that possess altered sensitivities to rapamycin (Xie et al., 2005). Intriguingly, mutants identified in this study represent several processes that have not been so far linked to TORC1 (Xie et al., 2005). Thus, the scope of cellular activities influenced by TORC1, although already extensive, could be even greater than is presently recognized.
Another important unresolved question concerns the site(s) of action of TORC1 and TORC2 within the cell. Previous studies have shown, using a variety of experimental approaches, that a significant portion of Tor1p and Tor2p are associated with an internal set of membranes that are adjacent to, yet apparently distinct from, the plasma membrane (Cardenas and Heitman, 1995; Kunz et al., 2000; Wedaman et al., 2003). In particular, using immunoelectron microscopy, we have localized both Tor1p and Tor2p as well as their common binding partner Lst8p to an intracellular membrane population that, at the ultrastructural level, closely resembles characteristic tracks and/or tubules that have been attributed to membranes of the endocytic pathway (Wedaman et al., 2003). By contrast, two components specific to TORC1, Tco89p and Kog1p, are also localized to vacuolar membranes, suggesting a more complex intracellular distribution of TORC1 components (Huh et al., 2003; Reinke et al., 2004; Araki et al., 2005). Moreover, it has recently been reported that a portion of Tor1p is localized within the nucleus where it specifically regulates 35S rDNA expression (Li et al., 2006). Thus, a full description of the intracellular environment(s) of the TOR kinases and their partners remains an important goal, and it is likely to be essential for a complete understanding of the functions of TORC1 and TORC2.
Here, we report findings that bear on several of these issues by characterizing further the membrane environment of TOR. We find that Tor1p and Tor2p associate with a novel form of detergent-resistant membranes that are enriched for proteins involved in actin cytoskeleton organization as well as endocytosis. Genetic analyses reveal a remarkable number of functional interactions between these proteins and components of TORC1; moreover, we demonstrate that, in addition to previously described effects on actin depolarization, rapamycin treatment severely impairs fluid phase endocytosis. Combining our genetic analyses with database mining, we constructed a network of functional interactions that identifies cellular pathways that are likely to be influenced by both TORC1 and actin/endocytosis-related components. This systems-based approach allowed us to establish a novel connection between TORC1 and a number of additional genes involved in vesicular trafficking.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Longtine et al., 1998; Reinke et al., 2004). RVS161, SLA2, SEC8, KOG1, TCO89, LST8, and AVO3 were tagged at their carboxy termini with multiple copies of the Myc epitope as described previously (Brachmann et al., 1998; Longtine et al., 1998; Reinke et al., 2004). TOR1, TOR2, and RSP5 were tagged at their amino termini with three copies of the hemagglutinin (HA) epitope as described previously (Brachmann et al., 1998; Longtine et al., 1998; Wedaman et al., 2003; Reinke et al., 2004). Rapamycin (LC Labs, Woburn, MA) was added to YPD medium when indicated at a final concentration of 0.2 µg/ml.
|
Triton X-100 (TX-100) Supernatant/Pellet Assay
For each strain tested, 320 ml of cells was grown overnight at 30°C to 0.5 OD600/ml in YPD (160 OD cells). Cells were pelleted in 50-ml conical tubes, washed in H2O, pelleted again, and resuspended at 40 OD cells/ml in TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) containing protease inhibitors (cocktail tablet; Roche Diagnostics) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Yeast cells were lysed by bead beating for 2 min. The lysates were cleared by centrifugation at 500 x g, for 5 min at 4°C. Cleared lysates were then spun at 20,000 x g for 20 min at 4°C, yielding 
2 ml of supernatant to be used for detergent treatment. After Bradford assays, the resulting supernatants were divided into 500-µl aliquots and treated 1:1 with TNE, TNE 1% Triton X-100, or TNE 1% Triton X-100 and 1 M NaCl (final concentrations). Samples were incubated on ice for 30 min during which time they were passed through a Hamilton syringe two times. Samples were spun at 100,000 x g for 1 h at 4°C. Supernatants were collected and trichloroacetic acid (TCA) precipitated, dried, and resuspended in 100 µl of sample buffer. Pellets were resuspended in 100 µl of sample buffer. Supernatants and pellets were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis.
OptiPrep Floatation Assay
OptiPrep floatation gradients and isolation of the Triton X-100–insoluble membranes were performed essentially as described previously (Bagnat et al., 2000) with minor modifications. Briefly, yeast cells were lysed by vortexing with glass beads in TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) supplied with protease inhibitors tablet (Roche Applied Science, Indianapolis, IN) and 1 mM PMSF (Sigma-Aldrich). The lysates were cleared by centrifugation at 500 x g for 5 min at 4°C, but they were not subjected to the 20,000 x g centrifugation step described for the Triton X-100 supernatant/pellet assay. Cleared lysates were incubated either with 1% TX-100 or with an equal volume of TNEX buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100) for 30 min on ice. The lysates were adjusted to 40% OptiPrep (Nycomed, Oslo, Norway), and 4.2 ml of resulting mixture was sequentially overlaid with 6.7 ml of 30% OptiPrep in TNEX buffer and 1.1 ml of TNEX buffer. The samples were centrifuged at 100,000 x g in SW41 Ti rotor for 2.5 h, and 1.2-ml fractions were collected from the top of the gradient and subjected to Western blot analysis.
Proteomic Analysis
TX-100–treated cell extracts were subjected to OptiPrep gradient as described above, and equivalent groups of fractions, corresponding to 0, 30, and 40% of OptiPrep, collected from four sets of the identical gradients, were combined. TOR-containing (30% OptiPrep) and plasma membrane raft (0% OptiPrep) fractions were repeatedly subjected to incubation with 1% TX-100 followed by floatation on an OptiPrep gradient as described above. Proteins from fractions corresponding to 0–30% OptiPrep of the second gradient as well as those corresponding to 40% of the first gradient were TCA precipitated and identified by tandem mass spectrometry as described previously (Carroll et al., 1998; Link et al., 1999).
Fluorescence Microscopy
Rhodamine-phalloidin staining of polymerized actin was performed as described previously (Pringle et al., 1989). For quantification of cells with depolarized actin cytoskeleton, 100 small-budded or midsize-budded cells were counted for each condition. Cells were considered as having depolarized actin patches if six or more patches were found within the mother cell. Lucifer yellow uptake assay was performed essentially as described previously (Dulic et al., 1991). Fluorescence microscopy was performed using Nikon E600 fluorescence microscope and Orca-ER charge-coupled device camera (Hamamatsu, Bridgewater, NJ) controlled by Simple PCI software (Compix, Cranberry, PA). All actin-staining images are z-series projections of optical sections.
Ste3-HA Endocytosis Assay
Monitoring of Ste3p-HA endocytosis was performed as described previously (Walther et al., 2006). Samples were diluted to give equal protein concentration determined using bicinchoninic acid assay and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting using anti-HA antibody.
CPY Trafficking Assay
Metabolic cell labeling and immunoprecipitations were performed essentially as described previously (Vashist et al., 2001). Briefly, 12 A600 OD units of early log phase cells were harvested and resuspended in 3.6 ml of SCD medium lacking methionine and cysteine. After 30 min of incubation at 30°C, cells were labeled with 500 µCi of [35S]methionine/cysteine (Promix; GE Healthcare), and chase was initiated by adding cold solution of methionine and cysteine (2 mM each in a final concentration) and terminated by the addition trichloroacetic acid to 10%. CPY was immunoprecipitated from cell lysates with the mAb specific to CPY and resolved by 8.5% SDS-PAGE. Quantification of CPY band images was performed using ImageQuant 5.0 software (GE Healthcare).
Subcellular Fractionation of Spheroplasts: Sorbitol Overlay Assay
Conversion of 250 OD cells to spheroplasts and cell lysis were performed as described previously (Harsay and Bretscher, 1995; Harsay and Schekman, 2002; Nunnari et al., 2002). For sorbitol density gradient centrifugation, 1 ml of cleared extract was overlaid directly onto sorbitol step gradients prepared in 12.5-ml tubes (SW41; Beckman Coulter, Fullerton, CA) as follows: 1 ml, 80%; 2 ml, 70%; 2 ml, 60%; 2 ml, 50%; 2 ml, 40%, and 1 ml, 30% sorbitol/1x YEB. The gradients were spun for 40 h at 200,000 x g, and 0.5-ml fractions were collected from the top. A portion of the material collected from each of the fractions was analyzed by SDS-PAGE and Western blot analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wedaman et al., 2003). We asked whether this was a general property of TORC1 and TORC2 by examining the behavior of representative components of each complex after detergent extraction and centrifugation of cell-free lysates. Indeed, for three members examined, Kog1p, Tco89p, and Avo3p, each behaved similarly to Tor1p and Tor2p in that they were efficiently converted to a soluble form only upon treatment with both 1% TX-100 and with 1 M NaCl (Figure 1). By contrast, a control membrane protein, the late endosomal marker Pep12p, was efficiently solubilized by detergent extraction alone (Figure 1).
|
; Simons and Vaz, 2004). One hallmark behavior of DRMs is their characteristic low buoyant density after detergent extraction and centrifugation in iodixanol (e.g., OptiPrep) step gradients (Bagnat et al., 2000). Thus, to test whether Tor1p and/or Tor2p are associated with these operationally defined DRMs, we adjusted both untreated as well as TX-100–treated cell extracts to 40% OptiPrep and resolved them on 0–40% OptiPrep discontinuous step gradients (see Materials and Methods). After centrifugation and fractionation, SDS-PAGE and Western blot analysis were used to determine the location of Tor1p and Tor2p as well as a number of different specific marker proteins (Figure 2, A and B).
|
), was localized at the top of gradients, corresponding to 0% OptiPrep fractions, in both untreated as well as in TX-100–treated extracts (Figure 2A). A similar behavior was also observed for another plasma membrane marker protein Chs3p (Ziman et al., 1996) (Figure 2A). By contrast, marker proteins for several distinct membrane compartments were observed to float in untreated extracts but not in TX-100–treated extracts, where in the latter case they largely cofractionated with the cytoplasmic marker protein Zwf1p (G6PDH) (Figure 2B). Interestingly, both Tor1p and Tor2p displayed a unique behavior, in comparison with each of the marker proteins described above, in that a portion of both proteins was spread throughout the 30% OptiPrep fractions and in a form that was largely resistant to TX-100 extraction (Figure 2A). Similar profiles were also observed for both Kog1p and Avo3p, components of TORC1 and TORC2, respectively (data not shown). These results demonstrate that a portion of both TORC1 and TORC2 are localized to a TX-100–resistant membrane environment that is distinct from DRMs that contained Pma1p and Chs3p. Accordingly, we differentiate between plasma membrane-DRMs (PM-DRMs) and TOR-DRMs to describe Pma1p/Chs3p-containing fractions versus Tor1p/Tor2p-containing fractions, respectively.
Sphingolipids are important for the formation and integrity of yeast DRMs (Simons and Vaz, 2004). Accordingly, we asked whether the TX-100-resistant behavior of Tor1p and Tor2p was altered in extracts prepared from a strain deleted for FEN1, which encodes fatty acid elongase required for normal levels of sphingolipids that contain very long chain fatty acids (Oh et al., 1997; Kohlwein et al., 2001; Obeid et al., 2002). We were particularly interested in comparing the behavior of the Tor proteins to Chs3p, which displayed an overlapping profile on OptiPrep gradients in the absence of TX-100 treatment (Figure 2A). We observed that the TX-100–resistant behavior of Chs3p was abolished in extracts prepared from a fen1
mutant (Figure 2C). By contrast, a significant amount of both Tor1p and Tor2p continued to float on OptiPrep gradients in detergent-treated extracts prepared from this mutant (Figure 2C), confirming the unique behavior of TOR-DRMs.
Proteomic Analysis of PM- and TOR-DRMs
To further characterize TOR-DRMs, we took advantage of its unique floatation profile after TX-100 treatment to isolate and identify cofractionating proteins in the upper 30% OptiPrep fractions using tandem MS/MS spectrometry (see Materials and Methods). For comparison, we also analyzed in parallel the protein composition of PM-DRMs from 0% OptiPrep fractions. Remarkably, the majority of proteins identified by mass spectrometry uniquely within PM-DRM fractions corresponded to known plasma membrane proteins, including a number of proteins that have previously been shown to reside in DRMs, such as Pma1p, Nce101p/Nce2p, Gas1p (Bagnat et al., 2000), and Sur7p (Malinska et al., 2004) as well as glycosylphosphatidylinositol-anchored proteins Ecm33p and Utr2p (Table 2).
|
|
; Reinke et al., 2004), whereas for TORC2 we examined deletions of two nonessential genes, AVO2 and BIT61 (Loewith et al., 2002; Reinke et al., 2004) as well as a novel temperature-sensitive allele of the essential gene AVO3 (see Materials and Methods). In each case, pairwise combinations of heterozygous diploid strains were constructed, followed by sporulation and tetrad dissection. We then assessed the degree of SSL interaction for each gene pair by genotyping the resulting spores and by inspection of individual spore sizes that formed at different temperatures.
From among the 60 heterozygous diploids strains examined, 10 synthetic lethal and eight synthetic slow-growth interactions were detected (summarized in Figure 3A). An example of both of these types of interactions is shown in Figure 3B, where simultaneous deletion of SLA2 and TOR1 results in synthetic lethality, whereas deletion of SLA2 and TCO89 results in a synthetic slow-growth phenotype. Some form of synthetic interaction was observed between one or more TORC1 or TORC2 components, and eight of the 10 genes tested from Table 3 (no detectable synthetic interactions were observed for RVS161 or SRO7). Four genes displayed synthetic interactions with both TORC1 as well as TORC2 components, whereas four genes displayed synthetic interactions exclusively with TORC1 components (Figure 3A). We note that the total number of interactions observed was significantly higher than predicted estimates of the total density of SSL interactions in the yeast genome (1:200; Tong et al., 2004), suggesting our proteomics approach led to an enrichment of gene products that interact functionally with TORC1 and TORC2.
|
). We confirmed this finding for two different wild-type strains, W303a and JK9-3da, where rapamycin treatment of cells for 30 min resulted in nearly complete depolarization of actin (Figure 4, A and B). Importantly, the budding index of cells did not change appreciably after drug treatment during this experiment, demonstrating that depolarization was not a secondary consequence of a rapamycin-induced cell cycle arrest, which remained a formal criticism of the previous study cited above (Figure 4C). Moreover, we observed significant depolarization after a period of rapamycin treatment as brief as 10 min (Figure 4D). By contrast, rapamycin treatment of cells expressing a dominant rapamycin resistant TOR1-1 allele had no significant effect on actin polarization, confirming that rapamycin-induced depolarization is due to inhibition of TORC1 (Figure 4, A and B).
|
). In this study, the kinetics of depolarization after glucose starvation was observed to be considerably faster than that caused by rapamycin treatment, leading to the conclusion that TOR was unlikely to mediate actin depolarization caused by glucose starvation (Uesono et al., 2004). However, a possible requirement for TOR during repolarization of the actin cytoskeleton was not addressed in this study. Given our present findings, we decided to test directly whether rapamycin treatment affected the kinetics of actin repolarization after glucose starvation. The results of this experiment are shown in Figure 5A.
|
). Significant repolarization was observed after 90 min of incubation in glucose-free media and nearly complete repolarization was observed at 120 min (Figure 5A). We note that this rate of repolarization was considerably faster than that reported previously, where it took approximate 5 h to significantly restore actin polarization (Uesono et al., 2004). We have determined that this difference is attributable to differences in strain background, where we used JK9–3da for this experiment, whereas the previous study used W303a (data not shown). Importantly, we found that addition of rapamycin to cells before a shift to glucose-free media strongly impeded the kinetics as well as extent of actin repolarization, demonstrating that a functional TORC1 is indeed required for actin remodeling in response to glucose starvation (Figure 5A). Given these results, we next wanted to ask whether there was a continued requirement for TOR once the actin cytoskeleton became repolarized after incubation in glucose-free media. We therefore starved cells for glucose for 120 min, followed by incubation for an additional 30 min either in the absence or presence of rapamycin (Figure 5B). We observed that the effect of rapamycin on actin depolarization was significantly attenuated, in comparison with an identical time of drug treatment of cells grown in YPD (Figure 5B, right). Thus, we conclude from these results that once actin becomes repolarized after glucose starvation, a requirement for TOR is much less critical for maintenance of polarity.
TORC1 Influences a Late Step of Endocytosis
Many of the genes that displayed SSL interactions with TORC1 components (Figure 3) have also been identified as being involved in receptor-mediated endocytosis (Kubler and Riezman, 1993; Wendland et al., 1996; Penalver et al., 1997; Wesp et al., 1997; deHart et al., 2003). We therefore examined the effect of rapamycin on the internalization of HA-epitope–tagged, GAL-regulated Ste3p, by using a galactose shut-off assay in conjunction with Western blot analysis to monitor protein turnover after internalization (Walther et al., 2006). No significant differences were observed in the rates of internalization within rapamycin-treated versus untreated cells, as judged by the disappearance of full-length Ste3p-HA after transfer of cells from galactose- to glucose-containing media (Figure 6A). This result is consistent with previous observations that rapamycin treatment does not impair 
factor internalization (deHart et al., 2003). However, we did observe a reproducible accumulation of faster mobility species of Ste3p-HA in rapamycin-treated cells (Figure 6A, top right, denoted by asterisks). These species most likely correspond to C-terminal fragments of Ste3p that have been observed previously to accumulate in mutants impaired for delivery of Ste3p to the vacuolar lumen (Chen and Davis, 2002; Shaw et al., 2003). As expected, appearance of these faster mobility intermediates required internalization of Ste3p-HA, because they did not occur in rapamycin-treated sla2 cells, where uptake of Ste3p is greatly diminished (Figure 6A).
|
). Here, we observed a significant delay in LY accumulation after rapamycin treatment, where only limited uptake was observed when cells were incubated with the dye for 60 min, a time corresponding to maximum uptake in untreated cells (Figure 5, B and C). A larger percentage of rapamycin-treated cells eventually displayed LY accumulation after an extended period of incubation with dye (90 min); however, the level of staining was always much more diffuse compared with untreated cells (Figure 6B). Together with our results with Ste3p-HA, we conclude that inhibition of TORC1 with rapamycin results in a significant delay in endocytosis but at a step that is likely to occur after internalization. By contrast to these results, rapamycin treatment resulted in no significant delay in the rate of accumulation of the lipophilic membrane marker FM4-64 at the vacuole, indicating that inhibition of TORC1 perturbs a limited scope of events related to endocytic trafficking to the vacuole (Figure 6C).
A genetic Network for TORC1 and Actin/Endocytosis Genes
Given our results described above, we sought to gain additional insight into the scope of cellular processes potentially influenced by both TORC1 and actin patch/endocytosis-related components. We explored a number of different approaches for meta-analysis, and we surveyed several interaction data sets available through the Saccharomyces Genome Database (SGD), and ultimately we devised the following regime to construct a network of genetic interactions (Figure 7). First, we defined our SSL interactions, described above, as LEVEL 1 of this network (Figure 7A) (Supplemental Table 2). Next, we identified through SGD 26 genes that display SSL interactions with at least two LEVEL 1 components and we defined these collectively as LEVEL 2 (Figure 7, B and C) (Supplemental Table 2). Not surprisingly, this list was enriched for genes implicated in actin organization (Figure 7C). We next identified through SGD 579 genes that display strictly synthetic lethal interactions with one or more LEVEL 2 genes, which we defined as LEVEL 3 (Supplemental Table 2). To narrow our focus to genes related specifically to TORC1, we filtered these LEVEL 3 genes through a list of 396 recently identified gene deletions that display altered (primarily increased) sensitivities to rapamycin (Xie et al., 2005) (Figure 7A). This latter step resulted in the identification of 108 genes that represent several distinct functional categories, as defined by the Gene Ontology index in SGD (Figure 7D and Supplemental Table 2).
|
. In particular, we wanted to determine whether our approach would ultimately link all 396 of these genes within the network, or, alternatively, whether this network would continue to identify only a subset of rapamycin responsive genes. Indeed, we observed that the network reached a plateau after the fifth LEVEL, and it included 250 rapamycin-responsive genes (Supplemental Table 2), which represented less than two thirds of the total number of genes identified by Xie et al. (2005). Additional functional categories derived from LEVELS 4 and 5 included processes linked to DNA metabolism as well as vacuole organization and biogenesis, autophagy, and cellular stress responses (Supplemental Table 2). Importantly, many processes commonly associated with TORC1 signaling, including carbon regulation as well as amino acid metabolism were not identified by this process, despite the fact that genes involved in these processes are included within the data set by Xie et al. (2005). Thus, these results suggest that the TORC1-actin/endocytosis network we have described involves a specific subset of rapamycin-responsive genes.
Testing the Network: Novel Genetic Interactions between TORC1 and Vesicular Trafficking Genes
A number of the processes listed in Figure 7D, including regulation of cell size, cell wall organization, and protein modification, have been shown previously to be regulated by TORC1. By contrast, several genes identified by this analysis cluster within the general category of "transport" and include genes that have not been linked previously to TORC1, except by virtue that their deletion results in hypersensitivity to rapamycin (Figure 7D) (Xie et al., 2005). Thus, to investigate this category further, we partitioned all genes within transport into distinct subcategories using the Gene Ontology index in SGD (Figure 8A). This approach identified three subcategories related to endosomal and vesicular transport, two of which possessed four genes in common: YPT6, VPS29, VPS35, and VPS38 (Figure 8A). Each of these genes has been implicated in the trafficking of a number of specific substrates, including CPY, which passes through the early secretory pathway and is targeted to the vacuole via the late endocytic pathway (Vashist et al., 2001). We therefore examined the effect of rapamycin treatment on the trafficking of CPY, which can be monitored using pulse-chase analysis as a series of post-translational glycosylation and proteolytic cleavage events as it transitions from two distinct precursor (P1 and P2) forms to a fully processed, mature (M) form. Indeed, we observed a mild but reproducible delay in CPY trafficking, particularly at the earliest step of conversion of P1 to P2 forms, in cells that had been treated with rapamycin, in comparison with untreated cells (Figure 8B). We note that this level of defect in CPY maturation is remarkably similar to that observed for cells deleted for the CHC1 gene that encodes clathrin heavy chain (Payne et al., 1988) as well as a number of mild mutant alleles of genes within the YPT gene family (Singer-Kruger et al., 1994).
|
and ypt6 as well as significant synthetic slow-growth interactions with each of the three vps gene deletions (Figure 8D). Together, these results demonstrate a remarkably tight series of functional interactions involving TORC1 and actin/endocytosis genes throughout this network.
Cofractionation of TORC1 with Distinct Organelle Markers
Given the spectrum of trafficking events that are predicted to be influenced by TORC1, both by our network analysis as well as results from other laboratories, we sought to revisit the question as to which intracellular membrane compartment(s) is associated with this complex. Results of previous studies are consistent with TORC1 components being localized to membranes related to the secretory and/or endosomal pathways (Kunz et al., 2000; Chen and Kaiser, 2003; Wedaman et al., 2003). However, as most biochemical fractionation approaches have used sucrose gradient floatation assays, it has been difficult to distinguish between these different compartments, due to the similar behavior of representative secretory and endosomal protein markers used for these assays. Accordingly, we explored a variety of gradient methods to maximize separation of different organelle markers from spheroplasted cell extracts, and we found that sorbitol overlay gradients (Cleves et al., 1991; McGee et al., 1994; Zinser and Daum, 1995) (see Materials and Methods) provided an effective means to distinguish several organelles, in particular markers for the trans-Golgi (Vps10p) versus the early endosome (Rsp5p) (Figure 9A).
|
; Reinke et al., 2004), displayed tight sedimentation profiles that most closely aligned with peak fractions for Rsp5p (Figure 9, A and B). By contrast, a significant portion of the TORC1 components Tor1p, Lst8p, and, to a lesser extent, Kog1p, showed a more diffuse sedimentation profile that was overlapping with several other organelle markers, including the trans-Golgi, endoplasmic reticulum (ER), and vacuole (Figure 9B). A control experiment using a Percoll gradient floatation assay confirmed that a significant portion of each TORC1 and TORC2 components examined behaved as a membrane-associated protein (Supplemental Figure 1). Moreover, subsequent analysis of two distinct Tor1p-containing fractions (7 and 15), taken from the sorbitol overlay gradient and used in an OptiPrep underlay assay, confirmed that a significant portion of Tor1p from each sorbitol overlay gradient fraction was membrane associated (our unpublished data). Together, these results are consistent with the conclusion that both TORC1 and TORC2 are likely to associate with an endosome-like compartment and that TORC1 associates with other compartments as well. This conclusion is consistent with our previous immunoelectron microscopy results where Tor2p displayed a more concise, punctate localization pattern adjacent to the plasma membrane, whereas Tor1p displayed a more dispersed localization pattern (Wedaman et al., 2003). Moreover, these results are consistent with previous findings that TORC1 components are also localized to the vacuole (Huh et al., 2003; Reinke et al., 2004; Araki et al., 2005). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Reinke et al., 2004), two processes previously ascribed strictly to TORC2, our results underscore the emerging connection with respect to cellular processes regulated by both TORC1 and TORC2 in yeast. This conclusion is consistent with recent findings for mammalian TOR signaling, where mTORC1 as well as mTORC2 have now been implicated in the regulation of polarized cell growth and/or cell movement as well as processes related to endocytosis (Berven et al., 2004; Jacinto et al., 2004; Sarbassov et al., 2004; Hennig et al., 2006). Moreover, these results are consistent with the unexpected degree of cross talk that has been demonstrated recently for these complexes in mammalian cells, where, for example, the AGC kinase family member PKB/Akt has been shown to act both downstream of mTORC2 as well as upstream of mTORC1 (Guertin and Sabatini, 2005; Sarbassov et al., 2005).
Based on our biochemical results, we distinguish detergent-resistant membranes that contain TOR from classically defined PM-DRMs that contain Pma1p as well as several other plasma membrane proteins, based on their differences in behavior on OptiPrep gradients as well as their response to perturbation of the sphingolipid content within cells. Cofractionation of proteins within TOR-DRMs could result from physical associations with either TORC1 or TORC2. For example, within TOR-DRMs, we identified Tpd3p, a structural component of the Pph21/22 phosphatase complex that is regulated by Tap42p and that acts downstream of TORC1 (Crespo and Hall, 2002). In this regard, Tap42p has recently been shown to interact directly with TORC1 within a detergent-resistant membrane fraction that is likely to correspond to TOR-DRMs, we have described here (Yan et al., 2006). Alternatively, cofractionation could be indicative of a shared biochemical environment that exists in the absence of direct physical interactions. In this regard, we have been unable to observe any significant association between Tor1p and Sla2p, as monitored by coimmunoprecipitation experiments (our unpublished data). Given the significant number of SSL interactions detected between TORC1 and components identified within TOR-DRMs, however, we suggest there are nevertheless significant functional links between proteins that cofractionate within TOR-DRMs.
Results of our genetic analyses, in combination with database mining, affirm the view that TORC1 affects a wide spectrum of cellular functions (Figure 7). This conclusion is further supported by our cell fractionation data that TORC1 may interact with a number of intracellular membrane compartments in addition to the plasma membrane (Figure 9). This latter conclusion is consistent with a recent report that yeast TORC1 is regulated by Pmr1p, a Golgi-localized Ca++/Mn++ ATPase (Devasahayam et al., 2006), as well as recent findings that mTOR interacts with both the ER as well as Golgi, in part due to the presence of specific sequences within N-terminal HEAT repeats (Liu and Zheng, 2007). Moreover, the utility of meta-analysis to extend our genetic interactions to construct an interaction network (Figure 7D) was demonstrated by the discovery of novel SSL interactions between TOR1 and components involved in intracellular trafficking (Figure 8). The interaction network we have described is based on a combination of SSL interactions as well as hypersensitivity of mutant strains to rapamycin (Xie et al., 2005). Thus, genes identified through this analysis may either represent components that function within the same pathway as TORC1, or, alternatively, within pathways that act in parallel to TORC1.
Previous studies have demonstrated that inhibition of TORC1 with rapamycin alters the intracellular trafficking of distinct nutrient-regulated amino acid permeases, including the tryptophan permease Tat2p and the general amino acid permease Gap1p, by altering their sorting at the trans-Golgi to the plasma membrane versus the vacuole (Schmidt et al., 1998; Chen and Kaiser, 2003). As these alterations mimic the physiological consequences of changes in amino acid and/or nitrogen source availability for these permeases, these results suggest that TORC1 is involved in modulating protein trafficking in response to nutritional signals (Wullschleger et al., 2006). Similarly, our findings reported here that rapamycin treatment significantly delays repolarization of the actin cytoskeleton after glucose starvation suggests TORC1 signaling may also be involved in cytoskeletal rearrangements that occur in response to changes in nutrient availability. According to this view, it is possible that other effects of rapamycin described here, including actin depolarization and delayed accumulation of lucifer yellow within the vacuole, are also in part consequences of complex cellular reorganization that occurs in response to nutrient deprivation in yeast. This notion is consistent with involvement of the actin cytoskeletal network in distinct membrane and protein trafficking events, many of which are important for cellular responses to starvation, including remodeling of the cell wall, as well as vacuole-related events during specific forms of autophagy (Hamasaki et al., 2005; Levin, 2005; Reggiori et al., 2005; He and Klionsky, 2007). Moreover, this view could account for the wide range of cellular processes that are coupled to both TORC1 and actin, as identified by our meta-analysis (Figure 7). Interestingly, TORC1 and components that regulate both the actin cytoskeleton as well as endocytosis are also linked in terms of the cell integrity response pathway, whereby plasma membrane and/or cell wall stability is modulated in response to thermal or osmotic stress (Torres et al., 2002; Reinke et al., 2004; Levin, 2005). These findings are consistent with proposals that there may be a common "regulatory architecture" whereby cells interpret and respond to nutritional as well as other environmental stresses (Levin, 2005). Our findings reported here underscore the important relationship between TORC1 and actin within this architecture.
|
ACKNOWLEDGMENTS |
|---|
-Pma1p and -Chs3p antibodies, P. Walter for -Sec61p antibodies and for plasmid pGAL-STE3-HA, and H. Pelham for plasmid pRS416-TLG1-MYC. We thank M. Madonna for establishing conditions for use of sorbitol overlay gradients and for generating preliminary results by using this assay. We thank K. Kaplan and the Kaplan laboratory for assistance with microscopy. This work was sponsored by National Science Foundation grant MCB-1031221 and American Cancer Society Research Scholar grant RSG-04-075-01-TBE (to T.P.). |
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Ted Powers (tpowers{at}ucdavis.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Audhya, A., Loewith, R., Parsons, A. B., Gao, L., Tabuchi, M., Zhou, H., Boone, C., Hall, M. N., and Emr, S. D. (2004). Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton. EMBO J. 23, 3747–3757.[CrossRef][Medline]
Bagnat, M., Keranen, S., Shevchenko, A., Shevchenko, A., and Simons, K. (2000). Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc. Natl. Acad. Sci. USA 97, 3254–3259.
Berven, L. A., Willard, F. S., and Crouch, M. F. (2004). Role of the p70(S6K) pathway in regulating the actin cytoskeleton and cell migration. Exp. Cell Res. 296, 183–195.[CrossRef][Medline]
Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.[CrossRef][Medline]
Cardenas, M., and Heitman, J. (1995). FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity. EMBO J. 14, 5892–5907.[Medline]
Carroll, C., Altman, R., Schieltz, D., Yates, J., and Kellogg, D. R. (1998). The septins are required for the mitosis-specific activation of the Gin4 kinase. J. Cell Biol. 143, 709–717.
Chen, E. J., and Kaiser, C. A. (2003). LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J. Cell Biol. 161, 1–15.
Chen, J. C., and Powers, T. (2006). Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae. Curr. Genet. 1–13.
Chen, L., and Davis, N. G. (2002). Ubiquitin-independent entry into the yeast recycling pathway. Traffic 3, 110–123.[CrossRef][Medline]
Cleves, A. E., McGee, T. P., Whitters, E. A., Champion, K. M., Aitken, J. R., Dowhan, W., Goebl, M., and Bankaitis, V. A. (1991). Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64, 789–800.[CrossRef][Medline]
Crespo, J. L., and Hall, M. N. (2002). Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 66, 579–591.
deHart, A.K.A., Schnell, J. D., Allen, D. A., Tsai, J.-Y., and Hicke, L. (2003). Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway. Mol. Biol. Cell 14, 4676–4684.
Devasahayam, G., Ritz, D., Helliwell, S. B., Burke, D. J., and Sturgill, T. W. (2006). Pmr1, a Golgi Ca2+/Mn2+-ATPase, is a regulator of the target of rapamycin (TOR) signaling pathway in yeast. Proc. Natl. Acad. Sci. USA 103, 17840–17845.
Di Como, C. J., and Arndt, K. T. (1996). Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 10, 1904–1916.
Dulic, V., Egerton, M., Elguindi, I., Raths, S., Singer, B., and Riezman, H. (1991). Yeast endocytosis assays. Methods Enzymol. 194, 697–710.[Medline]
Düvel, K., Santhanam, A., Garrett, S., Schneper, L., and Broach, J. R. (2003). Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. Mol. Cell 11, 1467–1478.[CrossRef][Medline]
Guertin, D. A., and Sabatini, D. M. (2005). An expanding role for mTOR in cancer. Trends Mol. Med. 11, 353–361.[CrossRef][Medline]
Hamasaki, M., Noda, T., Baba, M., and Ohsumi, Y. (2005). Starvation triggers the delivery of the endoplasmic reticulum to the vacuole via autophagy in yeast. Traffic 6, 56–65.[CrossRef][Medline]
Harsay, E., and Bretscher, A. (1995). Parallel secretory pathways to the cell surface in yeast. J. Cell Biol. 131, 297–310.
Harsay, E., and Schekman, R. (2002). A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J. Cell Biol. 156, 271–285.
He, C., and Klionsky, D. J. (2007). Atg9 trafficking in autophagy-related pathways. Autophagy 3, 271–274.[Medline]
Helliwell, S. B., Howald, I., Barbet, N., and Hall, M. N. (1998). TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics 148, 99–112.
Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R., and Hall, M. N. (1994). TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell 5, 105–118.[Abstract]
Hennig, K. M., Colombani, J., and Neufeld, T. P. (2006). TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J. Cell Biol. 173, 963–974.
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S., and O'Shea, E. K. (2003). Global analysis of protein localization in budding yeast. Nature 425, 681–691.[CrossRef][Medline]
Jacinto, E., Guo, B., Arndt, K. T., Schmelzle, T., and Hall, M. N. (2001). TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol. Cell 8, 1017–1026.[CrossRef][Medline]
Jacinto, E., Loewith, R., Schmidt, A., Lin, S., Ruegg, M. A., Hall, A., and Hall, M. N. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128.[CrossRef][Medline]
Jiang, Y., and Broach, J. R. (1999). Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 18, 2782–2792.[CrossRef][Medline]
Jorgensen, P., Rupes, I., Sharom, J. R., Schneper, L., Broach, J. R., and Tyers, M. (2004). A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev. 18, 2491–2505.
Kamada, Y., Fujioka, Y., Suzuki, N. N., Inagaki, F., Wullschleger, S., Loewith, R., Hall, M. N., and Ohsumi, Y. (2005). Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol. Cell. Biol. 25, 7239–7248.
Kohlwein, S. D., Eder, S., Oh, C. S., Martin, C. E., Gable, K., Bacikova, D., and Dunn, T. (2001). Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 109–125.
Kubler, E., and Riezman, H. (1993). Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 12, 2855–2862.[Medline]
Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. (1993). Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585–596.[CrossRef][Medline]
Kunz, J., Schneider, U., Howald, I., Schmidt, A., and Hall, M. N. (2000). HEAT repeats mediate plasma membrane localization of Tor2p in yeast. J. Biol. Chem. 275, 37011–37020.
Levin, D. E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69, 262–291.
Li, H., Tsang, C. K., Watkins, M., Bertram, P. G., and Zheng, X. F. (2006). Nutrient regulates Tor1 nuclear localization and association with rDNA promoter. Nature 442, 1058–1061.[CrossRef][Medline]
Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M., and Yates, J. R. (1999). Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.[CrossRef][Medline]
Liu, X., and Zheng, X. F. (2007). Endoplasmic reticulum and Golgi localization sequences for mammalian target of rapamycin. Mol. Biol. Cell 18, 1073–1082.
Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J. L., Bonenfant, D., Oppliger, W., Jenoe, P., and Hall, M. N. (2002). Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468.[CrossRef][Medline]
Longtine, M. S., McKenzie, A., III, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Malinska, K., Malinsky, J., Opekarova, M., and Tanner, W. (2004). Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells. J. Cell Sci. 117, 6031–6041.
Marion, R. M., Regev, A., Segal, E., Barash, Y., Koller, D., Friedman, N., and O'Shea, E. K. (2004). Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression. Proc. Natl. Acad. Sci. USA 101, 14315–14322.
McGee, T. P., Skinner, H. B., Whitters, E. A., Henry, S. A., and Bankaitis, V. A. (1994). A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes. J. Cell Biol. 124, 273–287.
Mulet, J. M., Martin, D. E., Loewith, R., and Hall, M. N. (2006). Mutual antagonism of target of rapamycin and calcineurin signaling. J. Biol. Chem. 281, 33000–33007.
Nasmyth, K., Adolf, G., Lydall, D., and Seddon, A. (1990). The identification of a second cell cycle control on the HO promoter in yeast: cell cycle regulation of SW15 nuclear entry. Cell 62, 631–647.[CrossRef][Medline]
Nunnari, J., Wong, E. D., Meeusen, S., and Wagner, J. A. (2002). Studying the behavior of mitochondria. Methods Enzymol 351, 381–393.[Medline]
Obeid, L. M., Okamoto, Y., and Mao, C. (2002). Yeast sphingolipids: metabolism and biology. Biochim. Biophys. Acta 1585, 163–171.[Medline]
Oh, C. S., Toke, D. A., Mandala, S., and Martin, C. E. (1997). ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J. Biol. Chem. 272, 17376–17384.
Payne, G. S., Baker, D., van Tuinen, E., and Schekman, R. (1988). Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast. J. Cell Biol. 106, 1453–1461.
Penalver, E., Ojeda, L., Moreno, E., and Lagunas, R. (1997). Role of the cytoskeleton in endocytosis of the yeast maltose transporter. Yeast 13, 541–549.[CrossRef][Medline]
Pringle, J. R., Preston, R. A., Adams, A. E., Stearns, T., Drubin, D. G., Haarer, B. K., and Jones, E. W. (1989). Fluorescence microscopy methods for yeast. Methods Cell Biol. 31, 357–435.[Medline]
Pruyne, D., and Bretscher, A. (2000). Polarization of cell growth in yeast. J. Cell Sci. 113, 571–585.[Abstract]
Reggiori, F., Monastyrska, I., Shintani, T., and Klionsky, D. J. (2005). The actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 16, 5843–5856.
Reinke, A., Anderson, S., McCaffery, J. M., Yates, J., 3rd, Aronova, S., Chu, S., Fairclough, S., Iverson, C., Wedaman, K. P., and Powers, T. (2004). TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J. Biol. Chem. 279, 14752–14762.
Rohde, J. R., and Cardenas, M. E. (2004). Nutrient signaling through TOR kinases controls gene expression and cellular differentiation in fungi. Curr. Top. Microbiol. Immunol. 279, 53–72.[Medline]
Sarbassov, D. D., Ali, S. M., Kim, D. H., Guertin, D. A., Latek, R. R., Erdjument-Bromage, H., Tempst, P., and Sabatini, D. M. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302.[CrossRef][Medline]
Sarbassov, D. D., Guertin, D. A., Ali, S. M., and Sabatini, D. M. (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101.
Schmelzle, T., Beck, T., Martin, D. E., and Hall, M. N. (2004). Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast. Mol. Cell. Biol. 24, 338–351.
Schmelzle, T., Helliwell, S. B., and Hall, M. N. (2002). Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol. Cell. Biol. 22, 1329–1339.
Schmidt, A., Beck, T., Koller, A., Kunz, J., and Hall, M. N. (1998). The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J. 17, 6924–6931.[CrossRef][Medline]
Schmidt, A., Bickle, M., Beck, T., and Hall, M. N. (1997). The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell 88, 531–542.[CrossRef][Medline]
Schmidt, A., Kunz, J., and Hall, M. N. (1996). TOR2 is required for organization of the actin cytoskeleton. Proc. Natl. Acad. Sci. USA 93, 13780–13785.
Shaw, J. D., Hama, H., Sohrabi, F., DeWald, D. B., and Wendland, B. (2003). PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body. Traffic 4, 479–490.[CrossRef][Medline]
Simons, K., and Vaz, W. L. (2004). Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33, 269–295.[CrossRef][Medline]
Singer-Kruger, B., Stenmark, H., Dusterhoft, A., Philippsen, P., Yoo, J. S., Gallwitz, D., and Zerial, M. (1994). Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J. Cell Biol. 125, 283–298.
Stamenova, S. D., Dunn, R., Adler, A. S., and Hicke, L. (2004). The Rsp5 ubiquitin ligase binds to and ubiquitinates members of the yeast CIN85-endophilin complex, Sla1-Rvs167. J. Biol. Chem. 279, 16017–16025.
Tabuchi, M., Audhya, A., Parsons, A. B., Boone, C., and Emr, S. D. (2006). The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation. Mol. Cell. Biol. 26, 5861–5875.
Tong, A. H. et al. (2004). Global mapping of the yeast genetic interaction network. Science 303, 808–813.
Torres, J., Di Como, C. J., Herrero, E., and Angeles de la Torre-Ruiz, M. (2002). Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast. J. Biol. Chem. 277, 43495–43502.
Uesono, Y., Ashe, M. P., and Toh, E. A. (2004). Simultaneous yet independent regulation of actin cytoskeletal organization and translation initiation by glucose in Saccharomyces cerevisiae. Mol. Biol. Cell 15, 1544–1556.
Vashist, S., Kim, W., Belden, W. J., Spear, E. D., Barlowe, C., and Ng, D. T. (2001). Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J. Cell Biol. 155, 355–368.
Vida, T. A., and Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792.
Walther, T. C., Brickner, J. H., Aguilar, P. S., Bernales, S., Pantoja, C., and Walter, P. (2006). Eisosomes mark static sites of endocytosis. Nature 439, 998–1003.[CrossRef][Medline]
Wang, H., and Jiang, Y. (2003). The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast. Mol. Cell. Biol. 23, 3116–3125.
Wedaman, K. P., Reinke, A., Anderson, S., Yates, J. I., McCaffery, J. M., and Powers, T. (2003). Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisae. Mol. Biol. Cell 14, 1204–1220.
Wendland, B., McCaffery, J. M., Xiao, Q., and Emr, S. D. (1996). A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J. Cell Biol. 135, 1485–1500.
Wesp, A., Hicke, L., Palecek, J., Lombardi, R., Aust, T., Munn, A. L., and Riezman, H. (1997). End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae. Mol. Biol. Cell 8, 2291–2306.
Wullschleger, S., Loewith, R., and Hall, M. N. (2006). TOR signaling in growth and metabolism. Cell 124, 471–484.[CrossRef][Medline]
Xie, M. W., Jin, F., Hwang, H., Hwang, S., Anand, V., Duncan, M. C., and Huang, J. (2005). Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. Proc. Natl. Acad. Sci. USA 102, 7215–7220.
Yan, G., Shen, X., and Jiang, Y. (2006). Rapamycin activates Tap42-associated phosphatases by abrogating their association with Tor complex 1. EMBO J. 25, 3546–3555.[CrossRef][Medline]
Ziman, M., Chuang, J. S., and Schekman, R. W. (1996). Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway. Mol. Biol. Cell 7, 1909–1919.[Abstract]
Zinser, E., and Daum, G. (1995). Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae. Yeast 11, 493–536.[CrossRef][Medline]
Zurita-Martinez, S. A., and Cardenas, M. E. (2005). Tor and cyclic AMP-protein kinase A: two parallel pathways regulating expression of genes required for cell growth. Eukaryot. Cell 4, 63–71.
This article has been cited by other articles:
|
|
 |
|
  E. Rolli, E. Ragni, J. Calderon, S. Porello, U. Fascio, and L. Popolo Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast Mol. Biol. Cell, November 15, 2009; 20(22): 4856 - 4870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Singh and M. Tyers A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis Genes & Dev., August 15, 2009; 23(16): 1944 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Frohlich, K. Moreira, P. S. Aguilar, N. C. Hubner, M. Mann, P. Walter, and T. C. Walther A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling J. Cell Biol., June 29, 2009; 185(7): 1227 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Georis, A. Feller, J. J. Tate, T. G. Cooper, and E. Dubois Nitrogen Catabolite Repression-Sensitive Transcription as a Readout of Tor Pathway Regulation: The Genetic Background, Reporter Gene and GATA Factor Assayed Determine the Outcomes Genetics, March 1, 2009; 181(3): 861 - 874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Berchtold and T. C. Walther TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain Mol. Biol. Cell, March 1, 2009; 20(5): 1565 - 1575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Wiederhold, T. Gandhi, H. P. Permentier, R. Breitling, B. Poolman, and D. J. Slotboom The Yeast Vacuolar Membrane Proteome Mol. Cell. Proteomics, February 1, 2009; 8(2): 380 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Hosiner, H. Lempiainen, W. Reiter, J. Urban, R. Loewith, G. Ammerer, R. Schweyen, D. Shore, and C. Schuller Arsenic Toxicity to Saccharomyces cerevisiae Is a Consequence of Inhibition of the TORC1 Kinase Combined with a Chronic Stress Response Mol. Biol. Cell, February 1, 2009; 20(3): 1048 - 1057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. W. Sturgill, A. Cohen, M. Diefenbacher, M. Trautwein, D. E. Martin, and M. N. Hall TOR1 and TOR2 Have Distinct Locations in Live Cells Eukaryot. Cell, October 1, 2008; 7(10): 1819 - 1830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Puria, S. A. Zurita-Martinez, and M. E. Cardenas From the Cover: Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae PNAS, May 20, 2008; 105(20): 7194 - 7199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Robinson and M. G. Buse Mechanisms of high-glucose/insulin-mediated desensitization of acute insulin-stimulated glucose transport and Akt activation Am J Physiol Endocrinol Metab, May 1, 2008; 294(5): E870 - E881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Diaz-Troya, F. J. Florencio, and J. L. Crespo Target of Rapamycin and LST8 Proteins Associate with Membranes from the Endoplasmic Reticulum in the Unicellular Green Alga Chlamydomonas reinhardtii Eukaryot. Cell, February 1, 2008; 7(2): 212 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Shen, C. S. Lancaster, B. Shi, H. Guo, P. Thimmaiah, and M.-A. Bjornsti TOR Signaling Is a Determinant of Cell Survival in Response to DNA Damage Mol. Cell. Biol., October 15, 2007; 27(20): 7007 - 7017. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
