Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Anticodon sequence determines the impact of mistranslating tRNAAla variants.

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

Tra1 controls the transcriptional landscape of the aging cell.

Gene expression undergoes considerable changes during the aging process. The mechanisms regulating the transcriptional response to cellular aging remain poorly understood. Here, we employ the budding yeast Saccharomyces cerevisiae to better understand how organisms adapt their transcriptome to promote longevity. Chronological lifespan assays in yeast measure the survival of nondividing cells at stationary phase over time, providing insights into the aging process of postmitotic cells. Tra1 is an essential component of both the yeast Spt-Ada-Gcn5 acetyltransferase/Spt-Ada-Gcn5 acetyltransferase-like and nucleosome acetyltransferase of H4 complexes, where it recruits these complexes to acetylate histones at targeted promoters. Importantly, Tra1 regulates the transcriptional response to multiple stresses. To evaluate the role of Tra1 in chronological aging, we took advantage of a previously characterized mutant allele that carries mutations in the TRA1 PI3K domain (tra1Q3). We found that loss of functions associated with tra1Q3 sensitizes cells to growth media acidification and shortens lifespan. Transcriptional profiling reveals that genes differentially regulated by Tra1 during the aging process are enriched for components of the response to stress. Notably, expression of catalases (CTA1, CTT1) involved in hydrogen peroxide detoxification decreases in chronologically aged tra1Q3 cells. Consequently, they display increased sensitivity to oxidative stress. tra1Q3 cells are unable to grow on glycerol indicating a defect in mitochondria function. Aged tra1Q3 cells also display reduced expression of peroxisomal genes, exhibit decreased numbers of peroxisomes, and cannot grow on media containing oleate. Thus, Tra1 emerges as an important regulator of longevity in yeast via multiple mechanisms.

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG.

Transfer RNA variants increase the frequency of mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, to frequencies approaching 10% in yeast and bacteria. Cells cope with these variants by having multiple copies of each tRNA isodecoder and through pathways that deal with proteotoxic stress. In this study, we define the genetic interactions of the gene encoding tRNASerUGG,G26A, which mistranslates serine at proline codons. Using a collection of yeast temperature-sensitive alleles, we identify negative synthetic genetic interactions between the mistranslating tRNA and 109 alleles representing 91 genes, with nearly half of the genes having roles in RNA processing or protein folding and turnover. By regulating tRNA expression, we then compare the strength of the negative genetic interaction for a subset of identified alleles under differing amounts of mistranslation. The frequency of mistranslation correlated with the impact on cell growth for all strains analyzed; however, there were notable differences in the extent of the synthetic interaction at different frequencies of mistranslation depending on the genetic background. For many of the strains, the extent of the negative interaction with tRNASerUGG,G26A was proportional to the frequency of mistranslation or only observed at intermediate or high frequencies. For others, the synthetic interaction was approximately equivalent at all frequencies of mistranslation. As humans contain similar mistranslating tRNAs, these results are important when analyzing the impact of tRNA variants on disease, where both the individual's genetic background and the expression of the mistranslating tRNA variant need to be considered.

The SAGA and NuA4 component Tra1 regulates Candida albicans drug resistance and pathogenesis.

Candida albicans is the most common cause of death from fungal infections. The emergence of resistant strains reducing the efficacy of first-line therapy with echinocandins, such as caspofungin calls for the identification of alternative therapeutic strategies. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase domain. In Saccharomyces cerevisiae, the assembly and function of SAGA and NuA4 are compromised by a Tra1 variant (Tra1Q3) with three arginine residues in the putative ATP-binding cleft changed to glutamine. Whole transcriptome analysis of the S. cerevisiae tra1Q3 strain highlights Tra1's role in global transcription, stress response, and cell wall integrity. As a result, tra1Q3 increases susceptibility to multiple stressors, including caspofungin. Moreover, the same tra1Q3 allele in the pathogenic yeast C. albicans causes similar phenotypes, suggesting that Tra1 broadly mediates the antifungal response across yeast species. Transcriptional profiling in C. albicans identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion, and filamentous growth. Indeed, the tra1Q3 allele reduces filamentation and other pathogenesis traits in C. albicans. Thus, Tra1 emerges as a promising therapeutic target for fungal infections.

Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription.

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II)-transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4- or TetR-VP16-activated promoters downstream of a mistranslating tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

The amino acid substitution affects cellular response to mistranslation.

Mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, occurs in all organisms. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. Interestingly, human genomes contain tRNA variants with the potential to mistranslate. Cells cope with increased mistranslation through multiple mechanisms, though high levels cause proteotoxic stress. The goal of this study was to compare the genetic interactions and the impact on transcriptome and cellular growth of two tRNA variants that mistranslate at a similar frequency but create different amino acid substitutions in Saccharomyces cerevisiae. One tRNA variant inserts alanine at proline codons whereas the other inserts serine for arginine. Both tRNAs decreased growth rate, with the effect being greater for arginine to serine than for proline to alanine. The tRNA that substituted serine for arginine resulted in a heat shock response. In contrast, heat shock response was minimal for proline to alanine substitution. Further demonstrating the significance of the amino acid substitution, transcriptome analysis identified unique up- and down-regulated genes in response to each mistranslating tRNA. Number and extent of negative synthetic genetic interactions also differed depending upon type of mistranslation. Based on the unique responses observed for these mistranslating tRNAs, we predict that the potential of mistranslation to exacerbate diseases caused by proteotoxic stress depends on the tRNA variant. Furthermore, based on their unique transcriptomes and genetic interactions, different naturally occurring mistranslating tRNAs have the potential to negatively influence specific diseases.

Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

A functional unfolded protein response is required for chronological aging in Saccharomyces cerevisiae.

Progressive impairment of proteostasis and accumulation of toxic misfolded proteins are associated with the cellular aging process. Here, we employed chronologically aged yeast cells to investigate how activation of the unfolded protein response (UPR) upon accumulation of misfolded proteins in the endoplasmic reticulum (ER) affects lifespan. We found that cells lacking a functional UPR display a significantly reduced chronological lifespan, which contrasts previous findings in models of replicative aging. We find exacerbated UPR activation in aged cells, indicating an increase in misfolded protein burden in the ER during the course of aging. We also observed that caloric restriction, which promotes longevity in various model organisms, extends lifespan of UPR-deficient strains. Similarly, aging in pH-buffered media extends lifespan, albeit independently of the UPR. Thus, our data support a role for caloric restriction and reduced acid stress in improving ER homeostasis during aging. Finally, we show that UPR-mediated upregulation of the ER chaperone Kar2 and functional ER-associated degradation (ERAD) are essential for proper aging. Our work documents the central role of secretory protein homeostasis in chronological aging in yeast and highlights that the requirement for a functional UPR can differ between post-mitotic and actively dividing eukaryotic cells.

Modulating Mistranslation Potential of tRNASer in Saccharomyces cerevisiae.

Transfer RNAs (tRNAs) read the genetic code, translating nucleic acid sequence into protein. For tRNASer the anticodon does not specify its aminoacylation. For this reason, mutations in the tRNASer anticodon can result in amino acid substitutions, a process called mistranslation. Previously, we found that tRNASer with a proline anticodon was lethal to cells. However, by incorporating secondary mutations into the tRNA, mistranslation was dampened to a nonlethal level. The goal of this work was to identify second-site substitutions in tRNASer that modulate mistranslation to different levels. Targeted changes to putative identity elements led to total loss of tRNA function or significantly impaired cell growth. However, through genetic selection, we identified 22 substitutions that allow nontoxic mistranslation. These secondary mutations are primarily in single-stranded regions or substitute G:U base pairs for Watson-Crick pairs. Many of the variants are more toxic at low temperature and upon impairing the rapid tRNA decay pathway. We suggest that the majority of the secondary mutations affect the stability of the tRNA in cells. The temperature sensitivity of the tRNAs allows conditional mistranslation. Proteomic analysis demonstrated that tRNASer variants mistranslate to different extents with diminished growth correlating with increased mistranslation. When combined with a secondary mutation, other anticodon substitutions allow serine mistranslation at additional nonserine codons. These mistranslating tRNAs have applications in synthetic biology, by creating "statistical proteins," which may display a wider range of activities or substrate specificities than the homogenous form.

Targeted sequencing reveals expanded genetic diversity of human transfer RNAs.

Transfer RNAs are required to translate genetic information into proteins as well as regulate other cellular processes. Nucleotide changes in tRNAs can result in loss or gain of function that impact the composition and fidelity of the proteome. Despite links between tRNA variation and disease, the importance of cytoplasmic tRNA variation has been overlooked. Using a custom capture panel, we sequenced 605 human tRNA-encoding genes from 84 individuals. We developed a bioinformatic pipeline that allows more accurate tRNA read mapping and identifies multiple polymorphisms occurring within the same variant. Our analysis identified 522 unique tRNA-encoding sequences that differed from the reference genome from 84 individuals. Each individual had ~66 tRNA variants including nine variants found in less than 5% of our sample group. Variants were identified throughout the tRNA structure with 17% predicted to enhance function. Eighteen anticodon mutants were identified including potentially mistranslating tRNAs; e.g., a tRNASer that decodes Phe codons. Similar engineered tRNA variants were previously shown to inhibit cell growth, increase apoptosis and induce the unfolded protein response in mammalian cell cultures and chick embryos. Our analysis shows that human tRNA variation has been underestimated. We conclude that the large number of tRNA genes provides a buffer enabling the emergence of variants, some of which could contribute to disease.

Sfp1 links TORC1 and cell growth regulation to the yeast SAGA-complex component Tra1 in response to polyQ proteotoxicity.

Chromatin remodeling regulates gene expression in response to the accumulation of misfolded polyQ proteins associated with Huntington's disease (HD). Tra1 is an essential component of both the SAGA/SLIK and NuA4 transcription co-activator complexes and is linked to multiple cellular processes, including protein trafficking and signaling pathways associated with misfolded protein stress. Cells with compromised Tra1 activity display phenotypes distinct from deletions encoding components of the SAGA and NuA4 complexes, indicating a potentially unique regulatory role of Tra1 in the cellular response to protein misfolding. Here, we employed a yeast model to define how the expression of toxic polyQ expansion proteins affects Tra1 expression and function. Expression of expanded polyQ proteins mimics deletion of SAGA/NuA4 components and results in growth defects under stress conditions. Moreover, deleting genes encoding SAGA and, to a lesser extent, NuA4 components exacerbates polyQ toxicity. Also, cells carrying a mutant Tra1 allele displayed increased sensitivity to polyQ toxicity. Interestingly, expression of polyQ proteins upregulated the expression of TRA1 and other genes encoding SAGA components, revealing a feedback mechanism aimed at maintaining Tra1 and SAGA functional integrity. Moreover, deleting the TORC1 (Target of Rapamycin) effector SFP1 abolished upregulation of TRA1 upon expression of polyQ proteins. While Sfp1 is known to adjust ribosome biogenesis and cell size in response to stress, we identified a new role for Sfp1 in the control of TRA1 expression, linking TORC1 and cell growth regulation to the SAGA acetyltransferase complex during misfolded protein stress.

Acceptor Stem Differences Contribute to Species-Specific Use of Yeast and Human tRNASer.

The molecular mechanisms of translation are highly conserved in all organisms indicative of a single evolutionary origin. This includes the molecular interactions of tRNAs with their cognate aminoacyl-tRNA synthetase, which must be precise to ensure the specificity of the process. For many tRNAs, the anticodon is a major component of the specificity. This is not the case for the aminoacylation of alanine and serine to their cognate tRNAs. Rather, aminoacylation relies on other features of the tRNA. For tRNASer, a key specificity feature is the variable arm, which is positioned between the anticodon arm and the T-arm. The variable arm is conserved from yeast to human. This work was initiated to determine if the structure/function of tRNASer has been conserved from Saccharomyces cerevisiae to human. We did this by detecting mistranslation in yeast cells with tRNASer derivatives having the UGA anticodon converted to UGG for proline. Despite being nearly identical in everything except the acceptor stem, human tRNASer is less active than yeast tRNASer. A chimeric tRNA with the human acceptor stem and other sequences from the yeast molecule acts similarly to the human tRNASer. The 3:70 base pair in the acceptor stem (C:G in yeast and A:U in humans) is a prime determinant of the specificity. Consistent with the functional difference of yeast and human tRNASer resulting from subtle changes in the specificity of their respective SerRS enzymes, the functionality of the human and chimeric tRNASerUGG molecules was enhanced when human SerRS was introduced into yeast. Residues in motif 2 of the aminoacylation domain of SerRS likely participated in the species-specific differences. Trp290 in yeast SerRS (Arg313 in humans) found in motif 2 is proximal to base 70 in models of the tRNA-synthetase interaction. Altering this motif 2 sequence of hSerRS to the yeast sequence decreases the activity of the human enzyme with human tRNASer, supporting the coadaptation of motif 2 loop⁻acceptor stem interactions.

The Pseudokinase Domain of Saccharomyces cerevisiae Tra1 Is Required for Nuclear Localization and Incorporation into the SAGA and NuA4 Complexes.

Tra1 is an essential component of the SAGA/SLIK and NuA4 complexes in S. cerevisiae, recruiting these co-activator complexes to specific promoters. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase (PI3K) domain. Unlike other PIKK family members (e.g., Tor1, Tor2, Mec1, Tel1), Tra1 has no demonstrable kinase activity. We identified three conserved arginine residues in Tra1 that reside proximal or within the cleft between the N- and C-terminal subdomains of the PI3K domain. To establish a function for Tra1's PI3K domain and specifically the cleft region, we characterized a tra1 allele where these three arginine residues are mutated to glutamine. The half-life of the Tra1[Formula: see text] protein is reduced but its steady state level is maintained at near wild-type levels by a transcriptional feedback mechanism. The tra1[Formula: see text] allele results in slow growth under stress and alters the expression of genes also regulated by other components of the SAGA complex. Tra1[Formula: see text] is less efficiently transported to the nucleus than the wild-type protein. Likely related to this, Tra1[Formula: see text] associates poorly with SAGA/SLIK and NuA4. The ratio of Spt7SLIK to Spt7SAGA increases in the tra1[Formula: see text] strain and truncated forms of Spt20 become apparent upon isolation of SAGA/SLIK. Intragenic suppressor mutations of tra1[Formula: see text] map to the cleft region further emphasizing its importance. We propose that the PI3K domain of Tra1 is directly or indirectly important for incorporating Tra1 into SAGA and NuA4 and thus the biosynthesis and/or stability of the intact complexes.

Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae.

Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiples fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP display its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington's disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.

Evolving Mistranslating tRNAs Through a Phenotypically Ambivalent Intermediate in Saccharomyces cerevisiae.

The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele (tti2-L187P) in Saccharomyces cerevisiae to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNASer containing a proline anticodon (UGG) is toxic, using our selection system we identified four tRNASerUGG variants, each with a single mutation, that mistranslate at a tolerable level. Mistranslating tRNALeuUGG variants were also obtained, demonstrating the generality of the approach. We characterized two of the tRNASerUGG variants. One contained a G26A mutation, which reduced cell growth to 70% of the wild-type rate, induced a heat shock response, and was lost in the absence of selection. The reduced toxicity of tRNASerUGG-G26A is likely through increased turnover of the tRNA, as lack of methylation at G26 leads to degradation via the rapid tRNA decay pathway. The second tRNASerUGG variant, with a G9A mutation, had minimal effect on cell growth, was relatively stable in cells, and gave rise to less of a heat shock response. In vitro, the G9A mutation decreases aminoacylation and affects folding of the tRNA. Notably, the G26A and G9A mutations were phenotypically neutral in the context of an otherwise wild-type tRNASer These experiments reveal a model for genetic code evolution in which tRNA anticodon mutations and mistranslation evolve through phenotypically ambivalent intermediates that reduce tRNA function.

Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response.

The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2 The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly.

The C-terminal residues of Saccharomyces cerevisiae Mec1 are required for its localization, stability, and function.

Mec1, a member of the phosphoinositide three-kinase-related kinase (PIKK) family of proteins, is involved in the response to replicative stress and DNA damage and in telomere maintenance. An essential 30 to 35 residue, the FATC domain is found at the C-terminus of all PIKK family members. To investigate the roles of the C-terminal residues of Mec1, we characterized alleles of Saccharomyces cerevisiae mec1 that alter the FATC domain. A change of the terminal tryptophan to alanine resulted in temperature-sensitive growth, sensitivity to hydroxyurea, and diminished kinase activity in vitro. Addition of a terminal glycine or deletion of one, two, or three residues resulted in loss of cell viability and kinase function. Each of these Mec1 derivatives was less stable than wild-type Mec1, eluted abnormally from a size exclusion column, and showed reduced nuclear localization. We identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A. The rpn3-L140P allele acted in a partially dominant fashion. It was not able to suppress the inviability of the C-terminal truncations or additions or the hydroxyurea sensitivity of mec1-W2368A. The rpn3-L140P allele restored Mec1-W2368A to nearly wild-type protein levels at 37°, an effect partially mimicked by the proteasome inhibitor MG-132. Our study supports a role for the C-terminus in Mec1 folding and stability, and suggests a role for the proteasome in regulating Mec1 levels.

Genetic evidence links the ASTRA protein chaperone component Tti2 to the SAGA transcription factor Tra1.

Tra1 is a 3744-residue component of the Saccharomyces cerevisiae SAGA, NuA4, and ASTRA complexes. Tra1 contains essential C-terminal PI3K and FATC domains, but unlike other PIKK (phosphoinositide three-kinase-related kinase) family members, lacks kinase activity. To analyze functions of the FATC domain, we selected for suppressors of tra1-F3744A, an allele that results in slow growth under numerous conditions of stress. Two alleles of TTI2, tti2-F328S and tti2-I336F, acted in a partially dominant fashion to suppress the growth-related phenotypes associated with tra1-F3744A as well as its resulting defects in transcription. tti2-F328S suppressed an additional FATC domain mutation (tra1-L3733A), but not a mutation in the PI3K domain or deletions of SAGA or NuA4 components. We find eGFP-tagged Tti2 distributed throughout the cell. Tti2 is a component of the ASTRA complex, and in mammalian cells associates with molecular chaperones in complex with Tti1 and Tel2. Consistent with this finding, Tra1 levels are reduced in a strain with a temperature-sensitive allele of tel2. Further agreeing with a possible role for Tti2 in the folding or stabilization of Tra1, tra1-F3744A was mislocalized to the cytoplasm, particularly under conditions of stress. Since an intragenic mutation of tra1-R3590I also suppressed F3744A, we propose that Tti2 is required for the folding/stability of the C-terminal FATC and PI3K domains of Tra1 into their functionally active form.

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1.

Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins.