Role of the Tsc1-Tsc2 Complex in Signaling and Transport Across the Cell Membrane in the Fission Yeast Schizosaccharomyces pombe

Corresponding author: Tomohiro Matsumoto, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan, 606-8501., tmatsumo{at}house.rbc.kyoto-u.ac.jp (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Heterozygous inactivation of either human TSC1 or TSC2 causes tuberous sclerosis (TSC), in which development of benign tumors, hamartomas, occurs via a two-hit mechanism. In this study, fission yeast genes homologous to TSC1 and TSC2 were identified, and their protein products were shown to physically interact like the human gene products. Strains lacking tsc1⁺ or tsc2⁺ were defective in uptake of nutrients from the environment. An amino acid permease, which is normally positioned on the plasma membrane, aggregated in the cytoplasm or was confined in vacuole-like structures in tsc1 and tsc2 strains. Deletion of tsc1⁺ or tsc2⁺ also caused a defect in conjugation. When a limited number of the cells were mixed, they conjugated poorly. The conjugation efficiency was improved by increased cell density. tsc1 cells were not responsive to a mating pheromone, P-factor, suggesting that Tsc1 has an important role in the signal cascade for conjugation. These results indicate that the fission yeast Tsc1-Tsc2 complex plays a role in the regulation of protein trafficking and suggest a similar function for the human proteins. We also show that fission yeast Int6 is involved in a similar process, but functions in an independent genetic pathway.

TUBEROUS sclerosis (TSC) is an autosomal dominant syndrome characterized by the widespread development of benign tumors termed hamartomas. TSC affects 1 in 5800 individuals and is characterized by a broad phenotypic spectrum including seizures, mental retardation, renal dysfunction, and dermatological abnormalities (KWIATKOWSKI and SHORT 1994 ; GOMEZ et al. 1999 ). Linkage and positional cloning studies led to identification of two responsible genes, TSC1 on chromosome 9q34 (VAN SLEGTENHORST et al. 1997 ) and TSC2 on chromosome 16p13 (EUROPEAN CHROMOSOME 16 TUBEROUS SCLEROSIS CONSORTIUM 1993). Germline mutations of these genes appear to inactivate gene function. Loss of heterozygosity at either of the two loci occurs in TSC tumors, thus indicating that TSC1 and TSC2 genes are tumor suppressors (CARBONARA et al. 1994 ; GREEN et al. 1994A , GREEN et al. 1994B ; HENSKE et al. 1996 ).

It has recently been reported that the mammalian TSC1 protein hamartin interacts with members of the ezrin-radixin-moesin family of actin-binding proteins and may have a role in cytoskeletal organization. Antibody-mediated inactivation of hamartin results in cell retraction, loss of focal adhesions, cell rounding, and detachment from the substrate (LAMB et al. 2000 ). These phenotypes suggest a possible role of the hamartin in maintenance of cell-matrix and cell-cell adhesions. However, a relatively mild defect in actin cytoskeletal organization was seen in Tsc1 null murine embryo fibroblasts (KWIATKOWSKI et al. 2002 ). A Drosophila gene homologous to human TSC2 is allelic to the gigas gene (ITO and RUBIN 1999 ), whose mutation results in the appearance of enlarged cells in wing and eye imaginal discs. Although the biochemical mechanism for the gigas phenotype is unknown, additional genetic studies suggest that dTsc1 and dTsc2 antagonize the insulin signaling pathway in Drosophila (GAO and PAN 2001 ; POTTER et al. 2001 ). Another study has suggested that the two Drosophila genes might be involved in regulation of the levels of multiple cyclins (TAPON et al. 2001 ). The TSC2 protein tuberin contains a domain that is homologous to a domain present in several proteins with GTPase-activating protein (GAP) activity for the ras family of GTPases. Biochemical studies have shown that tuberin has weak GAP activity for both rap1 and rab5 (WIENECKE et al. 1995 ; XIAO et al. 1997 ).

Thus, the cellular phenotypes associated with loss of either the TSC1 or the TSC2 gene in higher eukaryotes are complex and the biological function of the proteins remains poorly understood. Here we have approached this problem using fission yeast that appear to contain homologs of TSC1 and TSC2. We demonstrate that deletion of either gene causes a defect in transport signaling across the cell membrane. We also show that the phenotype is aggravated when combined with loss of int6⁺, a gene homologous to a target of MMTV (mouse mammary tumor virus).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
All strains used in this study were derived from wild-type strains. Schizosaccharomyces pombe was grown in standard yeast extracts plus adenine (YEA) and pombe minimum (PM) media (BEACH et al. 1985 ). The double mutants used in this study were constructed by tetrad analysis.

Cloning of tsc1⁺ and tsc2⁺:
The fission yeast tsc1⁺ gene was isolated from a cosmid clone (clone 541) that was previously mapped on the fission yeast genome (MIZUKAMI et al. 1993 ). A 4.6-kb PvuII-XhoI fragment that contained the complete open reading frame of tsc1⁺ was subcloned into pSP1 (COTTAREL et al. 1993 ) after the PvuII site was converted to a NotI site. The resulting plasmid was designated pSP1-Tsc1xn. Similarly, the tsc2⁺ gene was isolated from a cosmid (clone 630). A 6.9-kb ApaI-XhoI fragment that contained the complete open reading frame of tsc2⁺ was subcloned into pSP1 to generate pSP1-Tsc2.

Deletion and tagging:
For deletion of tsc1⁺, a 1.8-kb BamHI fragment that contained the complete open reading frame of ura4⁺ was inserted into the open reading frame of tsc1⁺. The position of the insertion was between a BglII site located 166 bp downstream of the first ATG and a BamHI site located 1147 bp downstream of the BglII site. For deletion of tsc2⁺, the 1.8-kb BamHI fragment containing ura4⁺ was inserted between a SnaBI (1042 bp downstream of the first ATG) and a SpeI site (8 bp upstream of the termination codon).

Green fluorescent protein (GFP) was tagged to the C terminus of Tsc1 as follows: A NotI site was generated immediately before the termination codon of tsc1⁺. A 1.1-kb Spe1-NotI fragment containing the C-terminal region of tsc1⁺ was subcloned into a plasmid (pYC11-6xGFP; gift from Dr. Yanagida). The plasmid contained the GFP gene that had a NotI site immediately before the first ATG for fusion with tsc1⁺. A haploid wild-type strain was transformed with the resulting plasmid, and stable transformants were examined for expression of Tsc1-GFP. Tsc1-GFP appeared as a doublet in both the immunoprecipitates (Fig 3, lanes 1 and 3) and the cell extracts (lanes 5 and 6). The protein may be modified although its nature is not known at present. Similarly, for tagging the GFP to the C terminus of the permease, NotI and SpeI sites were generated immediately before the termination codon and 0.85 kb upstream of the termination codon, respectively. A 0.85-kb Spe1-NotI fragment containing the C-terminal region of the permease was subcloned into a plasmid (pYC11-6xGFP, gift from Dr. Yanagida) and used for the integration.

View larger version (56K): In this window In a new window Download PPT slide   Figure 1. Comparison of Tsc1. The amino acid sequences of predicted fission yeast (sp) Tsc1 with its structural homologs from human (hs) and fly (dm) are aligned by the program DIALIGN (MORGENSTERN 1999 ). Two domains, which exhibit high homology, are shown in A and B. The alignments of the entire sequences are available upon request. The conserved residues between two or more species are highlighted. Potential transmembrane domains are boxed.

View larger version (64K): In this window In a new window Download PPT slide   Figure 2. Comparison of Tsc2. The amino acid sequences of predicted fission yeast (sp) Tsc2 with its structural homologs from human (hs) and fly (dm) are aligned by the program DIALIGN. Two domains, which exhibit high homology, are shown in A and B. The alignments of the entire sequences are available upon request. The conserved residues between two or more species are highlighted. Motifs characteristic of GAP are boxed.

View larger version (42K): In this window In a new window Download PPT slide   Figure 3. Physical interaction between Tsc1 and Tsc2. Tagged versions of Tsc1 and Tsc2 were expressed in fission yeast and were isolated using antibodies against the HA epitope (lanes 1 and 2) or GFP (lanes 3 and 4). After SDS-PAGE and membrane transfer, isolated proteins were probed with both antibodies. (Top) Anti-HA epitope. (Bottom) anti-GFP. The cell extracts were also examined by Western blot for the presence of Tsc1-GFP and Tsc2-HA. While the extracts used for the immunoprecipitation shown in lane 1 contained both Tsc1-GFP and Tsc2-HA (lane 5), the negative control extracts used for lane 2 contained Tsc1-GFP, but not Tsc2-HA (lane 6). Similarly, while the extracts used for the immunoprecipitation shown in lane 3 contained both Tsc1-GFP and Tsc2-HA (lane 5), the negative control extracts used for lane 4 contained Tsc2-HA, but not Tsc1-GFP (lane 7).

The hemagglutin (HA) epitope was tagged to the C terminus of Tsc2 as follows: A NotI fragment containing three repeats of the HA epitope was inserted into a NotI site that was generated on pSP1-Tsc2 immediately before the termination codon of tsc2⁺. The resulting plasmid, pSP1-Tsc2 C-HA, was digested by the restriction enzymes ApaI and XhoI, and the restriction fragment that contained tsc2⁺ tagged with the HA epitope was used to replace tsc2::ura4⁺.

Immunoprecipitation:
For immunoprecipitation (IP) and Western blotting, cell extracts were prepared by vortexing cells with glass beads in 1x PBS (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, and 8.1 mM Na₂HPO₄) containing 5 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate. The following proteinase inhibitors were added to the cell extracts: 0.1 mM phenylmethylsulfonyl fluoride, tosyl phenylalanyl chrolometyl ketone (10 µg/ml), leupeptin (2 µg/ml), aprotinin (2 µg/ml), and soybean trypsin inhibitor (10 µg/ml). For IP, total proteins of 3–5 mg in 1 ml were used. Western blot was performed with 200 µg of the total proteins for each lane.

Localization of permease and Map3:
The strains expressing the GFP-tagged permease, which were grown in the complete medium (YEA), were washed once with PM medium and grown in the same medium for 4 hr. The strains were fixed by methanol and observed under a fluorescent microscope. For study of localization of the pheromone receptor, Mps3, a multicopy plasmid (gift from Dr. Shimoda) containing the Mps3 coding region under control of the authentic promoter was transformed into h⁺ strains. The transformants, which were grown in PM, were transferred into PM lacking nitrogen for 1 hr, fixed by methanol, and observed under a fluorescent microscope.

Measurement of leucine uptake:
The method described previously (KARAGIANNIS et al. 1999 ) was followed with slight modification. Cells grown to the logarithmic phase in minimal medium, PM, were harvested and 1 ml of PM containing either 0.01 mM or 2 mM leucine supplemented with ³H-labeled leucine (2.5 µCi/ml, New England Nuclear, Boston) was added. The cells were incubated at 32° for the indicated time and mixed with chilled solution containing 100 mM cold leucine. They were immediately harvested, resuspended in the same solution, and filtered through GFA paper (0.45 µm, Whatman). The filter was washed with 1 ml of the same solution three times and subjected to a liquid scintillation spectrometer.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fission yeast tsc1⁺ and tsc2⁺:
Comparison of both human TSC1 (VAN SLEGTENHORST et al. 1997 ) and Drosophila TSC1 genes (ITO and RUBIN 1999 ) with the fission yeast genome permitted tentative identification of a homologous gene (accession no. S62428, hereafter termed fission yeast tsc1⁺). The fission yeast tsc1⁺ gene encodes a protein with a calculated molecular weight of 103 kD, which shares homologous domains with the human TSC1 (130 kD) and fly dTsc1 (125 kD), suggesting that they may have a similar biological function. In the first homologous domain (corresponding spTsc1: 175–474), fission yeast Tsc1 protein shows 27% identity to the human protein and 22% to the fly protein (Fig 1A). In the second domain (corresponding spTsc1: 545–604, Fig 1B), the fission yeast protein shows 36% identity to the human Tsc1. In addition, the three proteins are similar in overall structure. They contain a putative transmembrane domain at a conserved position (Fig 1A). In addition the C-terminal regions contain potential coiled-coil domains (predicted by Pairciol; BERGER et al. 1995 ; data not shown), although the precise locations and their primary sequences differ between the three species. The amino acid sequence of the fission yeast Tsc1 is shorter than those of the other two species. The human and fly counterparts contain an insertion (300 amino acids) that is not well conserved between the two species. Likewise, comparison of both human TSC2 (EUROPEAN CHROMOSOME 16 TUBEROUS SCLEROSIS CONSORTIUM, 1993) and Drosophila dTsc2 (ITO and RUBIN 1999 ; TAPON et al. 2001 ; GAO and PAN 2001 ; POTTER et al. 2001 ) genes identified a tentative homologous gene (accession no. CAB52735, hereafter termed fission yeast tsc2⁺) in the fission yeast genome. The amino acid sequence of the fission yeast Tsc2 (156 kD) is also shorter than those of the human and fly counterparts (196 and 204 kD, respectively). The N-terminal regions of the three genes are moderately conserved (Fig 2A). Furthermore, a domain near the C terminus (corresponding to spTsc2: 1066–1304) shows extensive homology (38% identity between human and fission yeast, Fig 2B). This domain contains a motif that is characteristic for GAP.

Since tuberin and hamartin, the products of the human TSC1 and TSC2 genes, physically interact with each other (PLANK et al. 1998 ; VAN SLEGTENHORST et al. 1998 ), we tested whether the fission yeast counterparts share this biochemical property. Fission yeast Tsc1 was tagged with GFP and Tsc2 with the HA epitope at their native loci. Immunoprecipitates using an antibody to the HA epitope contained both Tsc1-GFP and Tsc2-HA (Fig 3, lane 1). When Tsc2 was not tagged with the HA epitope, the precipitates did not contain Tsc1-GFP (lane 2), indicating specificity. In a reciprocal experiment, Tsc2-HA was precipitated with an antibody to GFP only when cell extracts contained Tsc1-GFP (lanes 3 and 4). The results demonstrated that fission yeast Tsc1 and Tsc2 form a complex. The fission yeast Tsc1 and Tsc2 appeared to be similar to the mammalian counterparts not only in structure but also in biochemical properties.

Defect in uptake:
Fission yeast strains with deletion of tsc1⁺ (tsc1) grew normally on complete medium YEA. When, however, tsc1 was combined with an auxotrophic marker, leu1-32, so that its growth depended on supplemental leucine, it required a higher concentration of leucine (Fig 4A). While the wild-type strain with leu1-32 grew normally with 50 µg/ml leucine on minimal medium, PM, tsc1 leu1-32 failed to grow under the same conditions. Even on PM medium supplemented with more leucine (200 µg/ml or 3 mg/ml), tsc1 leu1-32 grew more slowly than the single leu1-32 mutant. This defect was not observed in the single tsc1, indicating that deletion of tsc1⁺ does not affect growth on PM medium. Similarly, when tsc1 was combined with the ade6 auxotrophic marker, the double mutant tsc1 ade6 required supplemental adenine at a higher concentration (Fig 4B). We also combined tsc1 with other auxotrophic markers, lys1 and his3, and found that the double mutants required the corresponding supplemental amino acids at a higher concentration (not shown). Deletion of the tsc2⁺gene caused a similar defect. When combined with the leu1-32 or ade6 auxotrophic marker, tsc2 required the supplemental nutrients at a higher concentration (Fig 4A and Fig B). These results suggest that tsc1 and tsc2 are defective in uptake of nutrients from the environment.

View larger version (50K): In this window In a new window Download PPT slide   Figure 4. Growth defect of tsc1 and tsc2. Cells were suspended in liquid PM medium at a concentration of 5 x 104 cells/ml and 5 µl of the suspension was spotted on YEA or solid PM media containing leucine (A) or adenine (B) at concentrations indicated. They were incubated for 2 days (for YEA) or 5 days (for PM) at 32°. The genotype of each strain is indicated at the top.

To directly test tsc1 and tsc2 strains for their ability to take up nutrients, we measured uptake of ³H-labeled leucine. It has been shown previously that fission yeast utilizes two leucine-uptake systems, a high-affinity system with a substrate affinity (Kt) of 0.05 mM and a low-affinity system with a Kt of 1.25 mM (SYCHROVA et al. 1989 ; KARAGIANNIS et al. 1999 ). We first measured the uptake in the presence of 0.01 mM leucine so that only the high-affinity system would import leucine. As shown in Fig 5A, the uptake rate of tsc1 was 31% of the wild-type strain. Likewise, the rate of uptake by tsc2 was 35% of the wild type. Thus, in both tsc1 and tsc2, the high-affinity system for uptake of leucine is defective. We also measured the uptake in the presence of 2 mM leucine. The results (Fig 5B) indicate that the uptake rates of tsc1 and tsc2 were 37 and 53% of the wild-type strain, respectively. Although both the high- and low-affinity systems import leucine in the presence of 2 mM leucine, the efficiency of the low-affinity system is significantly higher than that of the high-affinity system (KARAGIANNIS et al. 1999 ). Therefore, the reduction in uptake is mostly attributable to a defect in the low-affinity system. These results indicate that both the high- and low-affinity systems for uptake of leucine are defective in tsc1 and tsc2, and to a similar extent.

View larger version (54K): In this window In a new window Download PPT slide   Figure 5. Defect in uptake and localization of permease. Uptake of radioactive-labeled leucine was measured in the presence of leucine at 0.01 mM (A) or at 2 mM (B). The vertical axes in A and B represent picomoles of leucine incorporated per 107 cells. The strain examined was the wild type (open circle), tsc1 (solid circle), tsc2 (solid triangle), int6 (open diamond), tsc1 int6 (solid square), and tsc2 int6 (open triangle). (C) Cells expressing the permease tagged with GFP were observed under a fluorescent microscope.

Localization of amino acid permease:
We reasoned that the defect in uptake could be caused if permeases, which are localized on the plasma membrane and actively import nutrients, are not fully functional. The fission yeast genome encodes at least six potential amino acid permeases. We selected one of them (accession no. CAB91572) and tagged GFP to its C terminus at the native locus. Analysis of the amino acid sequence of this potential permease indicated that: (1) It contains seven potential transmembrane domains and (2) it is highly homologous (37–39% identical) to other permeases (Gap1, general amino acid permease; Hip1, histidine permease; Tat2, tryptophan permease) that have been well characterized in budding yeast (TANAKA and FINK 1985 ; JAUNIAUX and GRENSON 1990 ; SCHMIDT et al. 1994 ).

In the wild-type strain, the GFP fluorescence was particularly bright along the edge of the cells, indicating that the majority of the permease was localized to the growing tips of fission yeast (Fig 5C). In contrast, the fluorescent signal was not detectable on the tips in tsc1. Instead, the GFP fluorescence was localized inside of the cells as patches near the tips. The observation suggests that the permease aggregates or is confined in vacuole-like structures in tsc1. In any case, the permease cannot be correctly placed on the plasma membrane in tsc1. The tsc2 cells exhibited abnormal distribution of the permease, which was similar to but somewhat milder than that in tsc1 (Fig 5C).

Genetic interaction between tsc1⁺ and tsc2⁺:
With the defect in uptake as a phenotypic marker, we examined genetic interaction between tsc1⁺ and tsc2⁺. First, tsc1 with the ade6-210 marker was transformed with a vector, a plasmid that allowed overexpression of tsc1⁺ (ptsc1+) or tsc2⁺ (ptsc2+). As shown in Fig 6A, the transformants with ptsc1+ were able to grow in the presence of a low concentration of adenine (50 µg/ml) on PM medium. The other transformants with the vector or ptsc2+ failed to do so. Similarly, tsc2 ade6-210 was rescued by ptsc2+, but not by ptsc1+. These results indicate that the defect associated with tsc1 or tsc2 is due to loss of function and that overexpression of one gene cannot substitute for the function of the other.

View larger version (34K): In this window In a new window Download PPT slide   Figure 6. Genetic interaction between tsc1+ and tsc2+. Cells were suspended in liquid PM medium at a concentration of 5 x 104 cells/ml and 5 µl of the suspension was spotted on solid PM medium containing adenine at concentrations indicated. They were incubated for 2 days at 32°. The genotype of each strain and plasmids used for transformation are indicated at the top.

Second, we combined tsc1 and tsc2 to test their interaction. As shown in Fig 6B, three strains, tsc1, tsc2, and tsc1 tsc2, exhibited similar defects in the uptake of adenine. On the basis of this result, we conclude that there is no additive interaction between tsc1 and tsc2. This genetic interaction, combined with the observation that Tsc1 and Tsc2 proteins form a complex, indicates that the products of the two genes function together in a step of a biological pathway.

Defect in conjugation:
Another defect found in tsc1 strains was their partial sterility, which was attributable to a low efficiency of conjugation. As shown in Table 1, tsc1 cells with opposite mating types (h⁺ and h^-) failed to conjugate when they were mixed at a low density. Under the same conditions, wild-type cells conjugated and succeeded in producing spores (Table 1). At a higher density, tsc1 conjugated and produced spores normally (Fig 8), suggesting that meiotic events occur normally in tsc1. As the conjugation efficiency was improved in a cell-density-dependent manner (Table 1), we suspected that tsc1 may not secrete the mating pheromone and/or may not respond to it efficiently. To further investigate the tsc1-phenotype, we examined the response of tsc1 to the mating pheromone, P-factor. P-factor, when added to nitrogen-starved cells with the h^- mating type, induces expression of a conjugation-specific gene, sxa2⁺ (IMAI and YAMAMOTO 1994 ). In response to the factor at 2 units/ml, the wild-type h^- cells expressed the sxa2⁺ gene at a detectable level (Fig 7). In contrast, h^- tsc1 was not responsive to the P-factor. Even with 50 units/ml of the pheromone, the sxa2 gene was not induced in tsc1 (Fig 7). Although we did not test directly, we assume that h⁺ tsc1 cells cannot respond to another mating pheromone, M-factor. The conjugation efficiency of the cross between h^- wild-type and h⁺ tsc1 strains was very similar to that between h⁺ wild-type and h^- tsc1 strains (Table 1). Thus, the conjugation defect associated with tsc1 is not specific to the mating type. It has also been shown that the receptors for P- and M-factors are similar in structure and that the signal is processed by the same biological pathway (DAVEY 1998 ). We also tested tsc2 strains for the ability to conjugate and found that they conjugated in a cell-density-dependent manner just like tsc1. Furthermore, tsc1 strains did not rescue the defect of tsc2 whereas the wild-type strains did (Table 1). Moreover, the conjugation efficiencies of wild type x tsc1 and wild type x tsc2 were very similar, as were the efficiencies of tsc1 x tsc1, tsc1 x tsc2, and tsc2 x tsc2, suggesting that these genes/proteins act in the same biochemical pathway.

View larger version (52K): In this window In a new window Download PPT slide   Figure 7. Response to P-factor. The wild-type (h-) cells or tsc1 (h-) cells were treated as described (IMAI and YAMAMOTO 1994 ) and total RNAs were examined by Northern blot with the full-length sxa2 gene as a probe. The amounts of the P-factor used are shown above each lane. rRNA stained with ethidium bromide, shown under each lane, proves loading of approximately the same amount of RNA.

View larger version (92K): In this window In a new window Download PPT slide   Figure 8. Genetic interaction of int6 with tsc1 and tsc2. (A) Growth defect. Cells were suspended in liquid PM medium at a concentration of 5 x 104 cells/ml and 5 µl of the suspension was spotted on YEA or solid PM media containing leucine at concentrations indicated. They were incubated for 2 days (for YEA) or 5 days (for PM) at 32°. The genotype of each strain is indicated at the top. (B) Fus- phenotype of tsc1 int6. Two strains (h+ and h-) were mixed at a concentration of 5 x 109 cells/ml and 5 µl of each cell suspension was spotted at a diameter of 5 mm on the meiosis-inducing medium and kept for 2 days at 26°. Genotypes are indicated at the top.

The pheromone produced by the opposite mating-type cells is recognized by cell surface receptors that activate the signal cascade for conjugation and meiosis (DAVEY 1998 ). The abnormal distribution of the amino acid permease in the tsc1 and tsc2 cells prompted us to hypothesize that localization of the pheromone receptors may also be affected. One of the receptors, Map3, was tagged with GFP and its localization was examined. As previously shown (HIROTA et al. 2001 ), the majority of Map3-GFP was found on the tips of the wild-type cells. Similarly, it was found on the tips in the tsc1 and tsc2 cells (not shown), suggesting that the defect in conjugation is not due to mislocalization of Map3 (see DISCUSSION).

Interaction with int6⁺:
Mouse Int6 locus is a target of MMTV. The infected mice frequently develop mammary tumors, which often result in metastatic lesions in the lung (MARCHETTI et al. 1995 ). It has also been shown that the Int6 protein associates with translation initiation factor 3, eIF3 (ASANO et al. 1997 ). The mechanism of the Int6-induced tumor and the role of the Int6 protein in translation initiation are still to be explored. We showed previously (BANDYOPADHYAY et al. 2000 , BANDYOPADHYAY et al. 2002 ) that deletion of fission yeast int6⁺, which is 43% identical to the mammalian counterpart, causes a number of phenotypes suggestive of a defect in the integrity of the cell membrane. In addition, it has been demonstrated that growth of int6 ade6-216 is reduced on PM (AKIYOSHI et al. 2001 ), suggesting that int6 might be defective in uptake as well.

In this study, we further analyzed the int6 phenotype in terms of a signaling/transport process across the cell membrane. First we tested whether the int6 cells could take up leucine normally. As shown in Fig 8A, a double mutant, int6 leu1-32, required leucine at a higher concentration. Consistently, measurement of uptake of ³H-labeled leucine (Fig 5A and Fig B) indicated that the rate of uptake by the int6 cells was 62% (high-affinity system) and 32% (low-affinity system) of the wild-type cells. To test whether Int6 plays a role in uptake in the same pathway with Tsc1 and Tsc2, we constructed double mutants, tsc1 int6 and tsc2 int6, and examined phenotypes. When combined with the leu1-32 marker, the double mutants were unable to grow on PM even with 3 mg/ml leucine added to the medium (Fig 8A). As shown in Fig 5A and Fig B, the combination of int6 and tsc1 or tsc2 aggravated the defect in uptake. The additive interaction would suggest that although Int6, Tsc1, and Tsc2 are required for uptake, Int6 plays a role that is independent from Tsc1 and Tsc2. Visualization of the permease GFP (Fig 5C) revealed that localization of the permease was abnormal in the int6 cells. Unlike the GFP signal in the wild-type cells that was mostly on the edge of the tips, the GFP fluorescence spread throughout inside the int6 cells. We also noted fluorescent patches inside, which were smaller and less bright than those found in the tsc1 or the tsc2 cells. The difference in the distribution of the permease also suggests that Int6 is required for uptake, but not for the same function as Tsc1 and Tsc2.

We next examined whether int6 additively interacts with tsc1 or tsc2 in conjugation. In contrast to tsc1 and tsc2, int6 conjugated normally at a low density although the homozygous cross of int6 often produced incomplete tetrads (Table 1, Fig 8B). Inactivation of both tsc1⁺ and int6⁺genes, however, caused a unique phenotype in conjugation. Under meiosis-inducing conditions, the wild-type cells elongate the conjugation tube. When the tube encounters that of the partner cell, the barrier between the two cells is removed. This event initiates fusion of cellular materials followed by completion of meiosis. As shown in Fig 8B, homozygous cross of double mutants (tsc1 int6) at high cell density failed to complete conjugation. The conjugation tubes were elongated and the two cells attached to each other. However, the barrier between the two cells remained intact. The conjugation phenotype of the double mutant (tsc1 int6) is similar to that seen in the fus1 mutant (PETERSEN et al. 1995 , PETERSEN et al. 1998 ) and is hence referred as the fus^- phenotype. The fus^- phenotype in the double mutant would suggest that Fus1 protein and/or its related proteins may not function correctly. We introduced a plasmid that would allow overexpression of the fus1⁺ gene from the ADH promoter (gift from Drs. Nielsen and Petersen) into the double mutant tsc1 int6. Fus1 overexpression did not suppress the fus^- phenotype of tsc1 int6 (S. MATSUMOTO and T. MATSUMOTO, unpublished result).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fission yeast tsc1⁺ and tsc2⁺:
On the basis of sequence similarity, we have identified putative homologs of human TSC1 and TSC2 genes in the fission yeast genome. The two proteins physically interact with each other in fission yeast, just as human proteins do. Therefore, we believe that the pair of tsc1⁺ and tsc2⁺ are functionally homologous to human TSC1 and TSC2. We have introduced the full-length human TSC1 into tsc1 and found that it did not rescue tsc1. The homology between the fission yeast and human genes is only moderate, so we suspect that their functions have diverged significantly, which explains this failure of cross-species complementation.

Despite an extensive search, we did not identify any homologs of TSC1 or TSC2 in the genome of budding yeast. The fission yeast model system we have discovered thus offers an opportunity to explore the functions of the Tsc1-Tsc2 complex by genetic approaches.

Defect in uptake:
When combined with auxotrophic markers such as leu1-32 and ade6-210, both tsc1 and tsc2 require supplemental nutrients at higher concentrations. We have also combined tsc1 with lys1 or his3. Although not extensively characterized, the growth rates of tsc1 lys1 and tsc1 his3 are reduced on the PM media supplemented with a standard concentration (50 µg/ml) of lysine and histidine, respectively. Therefore we propose that the fission yeast Tsc1-Tsc2 complex is required for uptake of various nutrients from the environment. Direct measurement of uptake of leucine supports this notion.

Uptake of a number of nutrients has been shown to be dependent on active transport. For example, uptake of most of the amino acids is dependent on specific transporters or permeases that are integrated into the plasma membrane and have transmembrane segments (reviewed in SOPHIANOPOULOU and DIALLINAS 1995 ). Prompted by the hypothesis that the receptors for amino acids may not be correctly positioned in tsc1 or tsc2, we have examined localization of one of the permeases. While the permease is homogeneously distributed on the growing tips of the wild-type cells, it was abnormally localized in tsc1 or tsc2 cells. Although the permeases were found near the tips, they were not displayed on the membrane. Instead, they were found as patches and perhaps inside of vacuole-like structures. It is also possible that the permease aggregates abnormally in the cytoplasm. Although we do not have direct evidence that this permease is the one that is responsible for the uptake defect present in tsc1 and tsc2, we suspect that localization of other permeases, most of which contain transmembrane domains and are believed to be membrane proteins, is also affected in tsc1 and tsc2. Abnormal distribution of these permeases would, at least in part, contribute to the defect in uptake.

Defect in conjugation:
tsc1 is not responsive to the P-factor. We speculate that this defect is the primary cause of the low efficiency of conjugation at a low cell density. Because the conjugation defect associated with tsc1 is not specific to the mating type, we suspect that it is not responsive to the M-factor as well. Unlike the amino acid permeases, the receptor for M-factor, Map3, seems to be positioned normally in tsc1 and tsc2 cells. We have also tried to localize the receptor for P-factor (Mam2), but found it technically difficult. At present, we have no evidence that suggests that the defect in conjugation is due to abnormal localization of the receptors, although it is still possible that the receptor is not functional even when positioned on the membrane. The pheromone signaling is mediated by a cascade that includes a cell surface receptor for the pheromone, a heterotrimeric G protein coupled to the receptor, and a mitogen-activated protein kinase module that can be activated by the G protein. As the final outcome, a transcriptional factor, Ste11, is stimulated upon the pheromone binding to the receptor (reviewed in DAVEY 1998 ). It has also been demonstrated that, upon binding of the pheromone, the receptor-pheromone complex is internalized (HIROTA et al. 2001 ; MORISHITA et al. 2002 ). Further experiments are necessary to elucidate which step in this process is affected in tsc1 and tsc2.

It should be noted that tsc1 may also be defective in production/secretion of pheromone. As shown in Table 1, the tsc1 sterile phenotype can be partially suppressed when mixed with the wild-type strain, suggesting that wild-type cells can induce the sexual differentiation in tsc1. Perhaps the wild-type cells can produce or secrete the pheromone at a level higher than that of tsc1. Therefore, the partial sterile phenotype observed in the cross of tsc1 x tsc1 at a low cell density could be due to defects in two processes, secretion/production of the mating pheromones and response to the pheromones.

Role of the Tsc1-Tsc2 complex:
Given the defects of tsc1 and tsc2 in uptake of nutrients and localization of the permease, we speculate that the fission yeast Tsc1-Tsc2 complex may play a role in correctly positioning the amino acid receptors. Receptor positioning requires multiple steps, including transcription and translation, post-translational processing, membrane integration, and cell surface targeting. The Tsc1-Tsc2 complex may be required for one or more such events. Our observation of Mps3-GFP in tsc1 and tsc2 indicates that localization of the pheromone receptor is not affected. The pheromone receptors may be positioned in a manner independent from Tsc1 and Tsc2. Thus, our results in aggregate of the permease suggest that Tsc1 and Tsc2 are involved in some membrane positioning events but not others.

Recent study in higher eukaryotes indicates the functional link between TSC2 and PKD1 (polycystin-1, a gene responsible for autosomal dominant polycystic kidney disease). In cells lacking the functional Tsc2, polycystin-1, which is normally localized on the membrane, is confined within Golgi apparatus (KLEYMENOVA et al. 2001 ). The Drosophila Tsc1-Tsc2 complex has been shown to regulate cell growth and proliferation via the insulin signaling pathway (POTTER et al. 2001 ; TAPON et al. 2001 ). This pathway would also require precise processing/transport of the receptors and their interacting proteins. Therefore, our findings on the role of the fission yeast Tsc1-Tsc2 complex are consistent with these reports. Tsc2 has been shown to act as GAP for the ras family of GTPases, Rap1 and Rab5, and Rab5 is known to be involved in cellular trafficking (WIENECKE et al. 1995 ; XIAO et al. 1997 ).

Interaction with int6⁺:
In the previous study (BANDYOPADHYAY et al. 2000 ), we showed that deletion of int6⁺ (int6) in fission yeast causes a defect in the integrity of the cell membrane. Because such a defect might affect a signaling/transport process, we tested genetic interaction with tsc1 and tsc2 in this study. int6 leu1-32 exhibits a growth defect on the PM medium supplemented with leucine at a low concentration, suggesting that it is also defective in uptake. Examination of the growth rates of the double mutants (int6 tsc1 and int6 tsc2) in the same test indicates that int6 additively interacts with tsc1 and tsc2. The additive interaction may indicate that Int6 serves an overlapping function with the Tsc1-Tsc2 complex. It should be noted that while int6 is hypersensitive to caffeine, tsc1 and tsc2 are not. Therefore, the overlapping function is only partial and the underlying mechanism of the gene function would be significantly different. We have also shown that int6 additively interacts with tsc1 in conjugation. The fus^- phenotype observed in the double mutant, int6 tsc1, suggests that the barrier between the cells cannot be removed. Removal of the barrier would require correct positioning of enzymes that degrade the cell wall. In the double mutant, such enzymes may not be functional.

Int6 is a subunit of eIF3, a translation initiation factor. Given the fact that the permease is not correctly positioned on the plasma membrane in cells lacking Int6, we speculate that Int6 may regulate synthesis of a subset of proteins specifically required for maintenance of the integrity of the cell membrane. It is equally possible that, in addition to its role in translation initiation, Int6 may play an additional role in a process for cellular trafficking or a related process.

Implication in human disease:
Keeping in mind that Tsc1, Tsc2, and Int6 in fission yeast are required for signaling/transport across the cell membrane, we propose that the human diseases, TSC and Int6-induced tumor, may be caused by a defect in a similar process. Control of growth and development in multiple organisms is, in part, mediated via signaling across the membrane. In many cases, binding of ligands and internalization of the receptor-ligand complex play a central role. Indeed, links between other proteins required for endocytosis and cancer have been postulated (FLOYD and DE CAMILLI 1998 ). Further studies in fission yeast should allow molecular dissection of Tsc and Int6 pathways and may provide crucial insights into the functions of the human homologs.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115.

	ACKNOWLEDGMENTS

We particularly thank Dr. Masayuki Yamamoto for his generous gift of the P-factor, helpful discussion, and comments on the manuscript. We also thank Drs. Nielsen, Petersen, Shimoda, and Yanagida for their gift of plasmids and strains. This research was supported by National Institutes of Health grants NS31535 (D.J.K.), GM15399 (U.M.), and GM56305 (T.M.) and the Tuberous Sclerosis Alliance (D.J.K.).

Manuscript received March 8, 2002; Accepted for publication May 2, 2002.

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