Involvement of Saccharomyces cerevisiae Avo3p/Tsc11p in maintaining TOR complex 2 integrity and coupling to downstream signaling

Abstract

Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated. Saccharomyces TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of avo3 temperature-sensitive (avo3(ts)) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by AVO1 while the other is suppressed by AVO2 or SLM1, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different avo3(ts) mutants. These molecular defects can be corrected only by expressing AVO3, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of avo3(ts) mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.

FIG. 1.

Interactions between Tor2p and other TORC2 components are differentially affected in different avo3^ts mutants. Mid-log-phase cultures of strains simultaneously expressing HA-tagged Tor2p and another, Myc-tagged TORC2 component were divided into two aliquots and further incubated at 27°C or 37°C for 2 h. Cell lysates were prepared and subjected to immunoprecipitation (IP) using anti-HA antibodies. Immunoprecipitates were separated by SDS-PAGE and examined for coimmunoprecipitated (CoIP) partner proteins by Western blot analysis using anti-Myc antibodies. Expression of the Myc-tagged TORC2 component in the lysates is shown at the bottom of each panel. “Bead control” (BC) samples were prepared using lysates from double-epitope-tagged wild-type (WT) strains without adding anti-HA antibodies during immunoprecipitation. (A) Tor2p-Avo1p interaction detected in YMY120 (WT), YMY218 (avo3-1^ts), and YMY318 (avo3-2^ts). (B) Tor2p-Avo2p interaction detected in YMY121 (WT), YMY219 (avo3-1^ts), and YMY319 (avo3-2^ts). (C) Tor2p-Avo3p interaction detected in YMY123 (WT), YMY221 (avo3-1^ts), and YMY321 (avo3-2^ts). (D) Tor2p-Lst8p interaction detected in YMY122 (WT), YMY220 (avo3-1^ts), and YMY320 (avo3-2^ts). (E) Schematic summary of TORC2 composition in different strains at the indicated temperatures. Shaded ovals indicate proteins with weakened interaction with Tor2p.

FIG. 2.

Interactions between TORC1 components are not affected in avo3^ts mutants. Strains expressing a Myc-tagged TORC1 component were subjected to the same procedures as those for Fig. 1. Immunoprecipitates were examined by Western blot analysis using anti-Tor1p antibodies. Expression of Tor1p in the lysates is shown at the bottom of each panel. WT, wild type; BC, bead control; IP, immunoprecipitation; CoIP, coimmunoprecipitation. (A) Tor1p-Lst8p interaction detected in YMY118 (WT), YMY216 (avo3-1^ts), and YMY316 (avo3-2^ts). (B) Tor1p-Kog1p interaction detected in YMY119 (WT), YMY217 (avo3-1^ts), and YMY317 (avo3-2^ts).

FIG. 3.

Overexpression of AVO3 but not allele-specific multicopy suppressors restores interactions between Tor2p and other TORC2 components in avo3^ts mutants. Plasmids expressing AVO3 (pTSS1), AVO1 (pHS2), AVO2 (pHS5), SLM1 (pHS6), or SLM2 (pHS12) and the control vector (pRS424) (Vec) were individually transformed into doubly tagged strains expressing HA-Tor2p and another, Myc-tagged TORC2 component. Lysates were prepared from transformants, and coimmunoprecipitation (CoIP) was performed as in the experiments for which results are shown in Fig. 1, by using anti-HA antibodies to immunoprecipitate (IP) HA-Tor2p and anti-Myc antibodies to detect the coprecipitated partner protein. Expression of the Myc-tagged TORC2 component in lysates is shown at the bottom of each panel. Overexp., overexpression; WT, wild type. (A) Tor2p-Avo1p interaction detected in transformants of YMY120 (WT) and YMY218 (avo3-1^ts). (B) Tor2p-Avo2p interaction detected in transformants of YMY121 (WT) and YMY219 (avo3-1^ts). (C) Tor2p-Avo3p interaction detected in transformants of YMY123 (WT) and YMY221 (avo3-1^ts). (D and H) Tor2p-Avo1p interaction detected in transformants of YMY120 (WT) and YMY318 (avo3-2^ts). (E and I) Tor2p-Avo2p interaction detected in transformants of YMY121 (WT) and YMY319 (avo3-2^ts). (F and J) Tor2p-Avo3p interaction detected in transformants of YMY123 (WT) and YMY321 (avo3-2^ts). (G) SLM2 serves as an avo3-2^ts-specific multicopy suppressor like SLM1. Transformants of YMY99 (avo3-1^ts) and YMY100 (avo3-2^ts) were subjected to a spot assay for temperature sensitivity. (K) Summary of the effects of individual avo3^ts mutant-specific suppressors on interactions between Tor2p and other TORC2 components. −, unable to rescue; +, able to rescue; ND, not determined.

FIG. 4.

Different avo3^ts mutations affect TORC2 interaction with Slm proteins differently. (A) Yeast strains were respectively transformed with plasmids to express GST (pGAL1-GST-URA3), GST-Slm1p (pHS10), or GST-Slm2p (pHS13) under the control of the GAL1 promoter. On the left, doubly tagged strains expressing 3HA-Tor2p and Avo2p-13Myc, including YMY121 (wild type [WT]), YMY219 (avo3-1^ts), and YMY319 (avo3-2^ts), were used. On the right, strains expressing 3HA-Tor2p, including YMY116 (WT), YMY215 (avo3-1^ts), and YMY315 (avo3-2^ts), were used. Transformants were induced to express SLM1 or SLM2 for 2 h and were further grown at 27°C or shifted to 37°C for another 2 h. Total lysates were prepared and subjected to GST pull-down procedures. The pulled down proteins were separated by SDS-PAGE; stained with Coomassie blue to visualize the pulled down GST, GST-Slm1p, or GST-Slm2p; and blotted with anti-HA or anti-Myc antibodies to detect the copurified 3HA-Tor2p or Avo2p-13Myc. Expression levels of 3HA-Tor2p and Avo2p-13Myc in lysates were also examined by Western blot analysis. (B) WT (YMY116) and avo2Δ (YMY124) strains expressing 3HA-Tor2p were respectively transformed with pGAL1-GST-URA3 or pHS10 and subjected to the same procedures as those for panel A. (C) YMY315 (avo3-2^ts) cells were respectively transformed with the control vector pRS424 (Vec), pHS5 (AVO2), or pTSS1 (AVO3), together with pHS10 or pHS13. The interaction of Tor2p with Slm1p or Slm2p was examined as described above and compared to that in YMY116 (WT) transformants.

FIG. 5.

The phenotypes of avo3^ts mutants can be partially rescued by deletion of specific RhoGAP-encoding genes. (A) Deletion of SAC7 partially rescues the temperature sensitivity of avo3^ts mutants. Tenfold serial dilutions of different yeast cultures were spotted onto YPD plates and incubated at different temperatures until colonies appeared. (B) The cell wall integrity of yeast strains carrying different GAP gene deletions was assessed by the trypan blue assay as described in Materials and Methods. The percentage of trypan blue-stained cells represents the extent of the cell wall defect of each strain. Shown are means ± standard deviations of results from three independent experiments. Statistical data were derived from Student's t test; each deletion strain was compared to its corresponding strain without any GAP gene deletion (No del). ***, P < 0.001; **, P < 0.005; *, P < 0.05. (C) Actin distribution in yeast strains carrying different GAP gene deletions was examined by TRITC-phalloidin staining and fluorescence microscopy. Small budded cells with four or more actin patches in the mother cell were counted as cells with abnormal actin distribution. Shown are means ± standard deviations of results from three independent experiments. Statistical data were obtained as for panel B.

FIG. 6.

Removal of the Sac7p GAP activity contributes to the rescue of avo3^ts mutants. (A) Comparison of GAP domains from different yeast RhoGAPs. The multiple sequence alignment was generated by T-Coffee analysis. The arrowhead indicates a conserved Arg residue at position 173 of Sac7p. (B) Mutation of Arg173 in Sac7p does not affect its Rho1p-binding activity. The interaction between Rho1p and Sac7p was examined using a GST pull-down assay. Glutathione Sepharose bead-bound GST or GST-Rho1p was incubated with yeast lysates containing HA-Sac7p or HA-Sac7p(R173A). Pulled down proteins were examined by Western blot analysis (WB) using anti-HA antibodies (upper panels) or Coomassie blue staining (lower panels). (C) Mutation of Arg173 in Sac7p affects its GAP activity. HA-Sac7p or HA-Sac7p(R173A) was incubated with [α-³²P]GTP-loaded GST-Rho1p to assay for GAP activity in vitro. Bound guanine nucleotides were extracted and analyzed by thin-layer chromatography. An autoradiograph of the thin-layer chromatographic plate is shown. (D) Spot assay for temperature sensitivity. Tenfold serial dilutions of the indicated yeast cell suspensions were spotted onto plates and incubated at different temperatures until colonies formed. Western blot analysis using anti-HA antibodies confirmed the expression of HA-Sac7p or HA-Sac7p(R173A) in strains.

FIG. 7.

Mutations of AVO3 cause decreases in active Rho1p (Act-Rho1) levels in cells. Lysates were prepared from transformants of YMY97 (wild type [WT]), YMY99 (avo3-1^ts), and YMY100 (avo3-2^ts) induced to express Rho1p-HA at 37°C for 2 h. The GTP-bound form of Rho1p-HA in lysates was pulled down using either GST-RBD (A) or GST-Pkc1-RBD (B). Pulled down samples were separated by SDS-PAGE; GST fusion proteins were detected by Coomassie blue staining, and active Rho1p-HA was detected by Western blot analysis using anti-HA antibodies. Total Rho1p-HA levels in lysates were also examined by Western blot analysis. Overexp., overexpression; Vec, vector.

FIG. 8.

Tus1p participates in Avo2p- and Slm1p-mediated, but not Avo1p-mediated, suppression of avo3^ts phenotypes. Three known Rho1p GEF genes, ROM1, ROM2, and TUS1, were individually deleted in avo3^ts mutants. The resulting strains were transformed by allele-specific multicopy suppressors. Transformants were subjected to spot assays for growth to test the effects of GEF deletion on suppression. (A) Rho1p-GEF deletions have no effect on AVO1-mediated suppression of the avo3-1^ts phenotype. Plasmid pRS424 (Vector), pTSS1 (AVO3), or pHS2 (AVO1) was transformed into YMY99 (avo3-1^ts), YMY212 (avo3-1^ts rom1Δ), YMY213 (avo3-1^ts rom2Δ), or YMY214 (avo3-1^ts tus1Δ). The resulting transformants were tested for growth at different temperatures. (B) Deletion of TUS1, but not ROM1 or ROM2, reduces AVO2- and SLM1-mediated suppression of the avo3-2^ts phenotype. Plasmid pRS424 (Vector), pTSS1 (AVO3), pHS5 (AVO2), or pHS6 (SLM1) was transformed into YMY100 (avo3-2^ts), YMY312 (avo3-2^ts rom1Δ), YMY313 (avo3-2^ts rom2Δ), or YMY314 (avo3-2^ts tus1Δ). Transformants were tested for growth at different temperatures.

FIG. 9.

Tus1p links Avo2p and Slm1p signaling to Rho1p activation. (A) Overexpression (Overexp.) of AVO2 or SLM1 restores levels of active Rho1p in avo3-2^ts cells. Plasmids expressing AVO3 (pTSS1), AVO2 (pHS5), or SLM1 (pHS6) from their endogenous promoters and the control vector (pRS424) were individually transformed into YMY100 (avo3-2^ts) or YMY314 (avo3-2^ts tus1Δ). After induction of Rho1p-HA expression at 37°C for 2 h, lysates were prepared and subjected to the pull-down assay for active Rho1p-HA (Act-Rho1) by using GST-Pkc1-RBD. Coomassie blue staining was used to detect GST-Pkc1p-RBD, while Western blot analysis with anti-HA antibodies was used to detect active and total Rho1p-HA. (B) Slm1p physically interacts with Tus1p. YMY97 (wild type [WT]) cells were cotransformed with plasmids expressing Tus1p-HA (pHS11) and GST (pGAL1-GST-URA3) or GST-Slm1p (pHS10) from the GAL1 promoter. Lysates were prepared from transformants induced by galactose and grown at 27°C or shifted to 37°C for 2 h; then the lysates were subjected to GST pull-down procedures. Pulled down proteins were separated by SDS-PAGE, stained by Coomassie blue to visualize the GST or GST-Slm1p, and examined by Western blot analysis with anti-HA antibodies for copurified Tus1p-HA. Expression levels of Tus1p-HA in lysates were also examined by Western blot analysis.