DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair

The yeast Mlh1–Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.     

The Unstructured Linker Arms of Mlh1-Pms1 Are Important for Interactions with DNA during Mismatch Repair

DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.     

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

Modeling of the DNA-binding site of yeast Pms1 by mass spectrometry

Mismatch repair (MMR) corrects replication errors that would otherwise lead to mutations and, potentially, various forms of cancer. Among several proteins required for eukaryotic MMR, MutLα is a heterodimer comprised of Mlh1 and Pms1. The two proteins dimerize along their C-terminal domains (CTDs), and the CTD of Pms1 houses a latent endonuclease that is required for MMR. The highly conserved N-terminal domains (NTDs) independently bind DNA and possess ATPase active sites. Here we use two protein footprinting techniques, limited proteolysis and oxidative surface mapping, coupled with mass spectrometry to identify amino acids involved along the DNA-binding surface of the Pms1-NTD. Limited proteolysis experiments elucidated several basic residues that were protected in the presence of DNA, while oxidative surface mapping revealed one residue that is uniquely protected from oxidation. Furthermore, additional amino acids distributed throughout the Pms1-NTD were protected from oxidation either in the presence of a non-hydrolyzable analog of ATP or DNA, indicating that each ligand stabilizes the protein in a similar conformation. Based on the recently published X-ray crystal structure of yeast Pms1-NTD, a model of the Pms1-NTD/DNA complex was generated using the mass spectrometric data as constraints. The proposed model defines the DNA-binding interface along a positively charged groove of the Pms1-NTD and complements prior mutagenesis studies of Escherichia coli and eukaryotic MutL.     

Functional domains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatch repair proteins and their relevance to human hereditary nonpolyposis colorectal cancer-associated mutations.

The MutL protein is an essential component of the Escherichia coli methyl-directed mismatch repair system but has no known enzymatic function. In the yeast Saccharomyces cerevisiae, the MutL equivalent, an Mlh1p and Pms1p heterodimer, interacts with Msh2p bound to mismatch-containing DNA. Little is known of the functional domains of Mlh1p and Pms1p. In this report, we define the Mlh1p and Pms1p domains required for Mlh1p-Pms1p interaction. The Mlh1p-interactive domain of Pms1p is comprised of 260 amino acids near the carboxyl terminus while the Pms1p-interactive domain of Mlh1p resides in the final 212 residues. The two domains are sufficient for Mlh1p-Pms1p interaction, as determined by the two-hybrid assay and by in vitro protein affinity chromatography. Deletions within the domains completely eliminated Mlh1p-Pms1p interaction. Using site-directed mutagenesis, we altered a number of highly conserved residues in the Mlh1p and Pms1p proteins, including some alterations that mimic germline mutations observed for human hereditary nonpolyposis colorectal cancer. Alterations either in the consensus MutL box located in the amino-terminal portion of each protein or in the carboxyl-terminal homology motif of Mlh1p eliminated DNA mismatch repair function but had no effect on Mlh1p-Pms1p interaction. In addition, certain MLH1 and PMS1 mutant alleles caused a dominant negative mutator effect when overexpressed. We discuss the implications of these findings for the structural organization of the Mlh1p and Pms1p proteins and the importance of Mlh1p-Pms1p interaction.     

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.     

Conservation of functional asymmetry in the mammalian MutLα ATPase

The DNA mismatch repair (MMR) protein dimer MutLα is comprised of the MutL homologues MLH1 and PMS2, which each belong to the family of GHL ATPases. These ATPases undergo functionally important conformational changes, including dimerization of the NH₂-termini associated with ATP binding and hydrolysis. Previous studies in yeast and biochemical studies with the mammalian proteins established the importance of the MutLα ATPase for overall MMR function. Additionally, the studies in yeast demonstrated a functional asymmetry between the contributions of the Mlh1 and Pms1 ATPase domains to MMR that was not reflected in the biochemical studies. We investigated the effect of mutating the highly conserved ATP hydrolysis and Mg²⁺ binding residues of MLH1 and PMS2 in mammalian cell lines. Amino acid substitutions in MLH1 intended to impact either ATP binding or hydrolysis disabled MMR, as measured by instability at microsatellite sequences, to an extent similar to MLH1-null mutation. Furthermore, cells expressing these MLH1 mutations exhibited resistance to the MMR-dependent cytotoxic effect of 6-thioguanine (6-TG). In contrast, ATP hydrolysis and binding mutants of PMS2 displayed no measurable increase in microsatellite instability or resistance to 6-TG. Our findings suggest that, in vivo, the integrity of the MLH1 ATPase domain is more critical than the PMS2 ATPase domain for normal MMR functions. These in vivo results are in contrast to results obtained previously in vitro that showed no functional asymmetry within the MutLα ATPase, highlighting the differences between in vivo and in vitro systems.     

Functional residues on the surface of the N-terminal domain of yeast Pms1

Saccharomyces cerevisiae MutLα is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5 Å crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.     

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg²⁺ and Mn²⁺ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.     

Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

The Huntington''s disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington''s disease Hdh^Q111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh^Q111) than on a 129 background (129.Hdh^Q111). Linkage mapping in (B6x129).Hdh^Q111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh^Q111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh^Q111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.     

Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.     

An intact Pms2 ATPase domain is not essential for male fertility

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2^ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2^ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2^cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2^cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2^cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2^ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2^cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3.     

Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1 proteins.

MutL homologs belong to a family of proteins that share a conserved ATP binding site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1 and Pms1 required for DNA mismatch repair both possess independent, intrinsic ATPase activities. Amino acid substitutions in the conserved ATP binding sites concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in vivo. The ATPase activities are weak, consistent with the hypothesis that ATP binding is primarily responsible for modulating interactions with other MMR components. Three approaches, ATP hydrolysis assays, limited proteolysis protection, and equilibrium dialysis, provide evidence that the amino-terminal domain of Mlh1 binds ATP with >10-fold higher affinity than does the amino-terminal domain of Pms1. This is consistent with a model wherein ATP may first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and later steps in DNA mismatch repair.     

Sensitivity to phosphonoacetic acid: a new phenotype to probe DNA polymerase delta in Saccharomyces cerevisiae

A mutant allele (pol3-L612M) of the DNA polymerase delta gene in Saccharomyces cerevisiae that confers sensitivity to the antiviral drug phosphonoacetic acid (PAA) was constructed. We report that PAA-sensitivity tagging DNA polymerases is a useful method for selectively and reversibly inhibiting one type of DNA polymerase. Our initial studies reveal that replication by the L612M-DNA pol delta requires Rad27 flap endonuclease activity since the pol3-L612M strain is not viable in the absence of RAD27 function. The L612M-DNA pol delta also strongly depends on mismatch repair (MMR). Reduced viability is observed in the absence of any of the core MMR proteins-Msh2, Mlh1, or Pms1-and severe sensitivity to PAA is observed in the absence of the core proteins Msh6 or Exo1, but not Msh3. We propose that pol3-L612M cells need the Rad27 flap endonuclease and MMR complexes composed of Msh2/Msh6, Mlh1/Pms1, and Exo1 for correct processing of Okazaki fragments.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.