Intragenic Suppressor Research Articles

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli.

The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection.

Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae.

The isolation of mutations affecting the stabilities of transmembrane proteins is useful for enhancing the suitability of proteins for structural characterization and identification of determinants of membrane protein stability. We have pursued a strategy for the identification of stabilized variants of the yeast α-factor receptor Ste2p. Because it was not possible to screen directly for mutations providing thermal stabilization, we first isolated a battery of destabilized temperature-sensitive variants, based on loss of signaling function and decreased levels of binding of the fluorescent ligand, and then screened for intragenic second-site suppressors of these phenotypes. The initial screens recovered singly and multiply substituted mutations conferring temperature sensitivity throughout the predicted transmembrane helices of the receptor. All of the singly substituted variants exhibit decreases in cell-surface expression. We then screened randomly mutagenized libraries of clones expressing temperature-sensitive variants for second-site suppressors that restore elevated levels of binding sites for fluorescent ligand. To determine whether any of these were global suppressors, and thus likely stabilizing mutations, they were combined with different temperature-sensitive mutations. Eight of the suppressors exhibited the ability to reverse the defect in ligand binding of multiple temperature-sensitive mutations. Combining certain suppressors into a single allele resulted in levels of suppression greater than that seen with either suppressor alone. Solubilized receptors containing suppressor mutations in the absence of temperature-sensitive mutations exhibit a reduced tendency to aggregate during immobilization on an affinity matrix. Several of the suppressors also exhibit allele-specific behavior indicative of specific intramolecular interactions in the receptor.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface.

In Escherichia coli, initial assembly of the Z ring for cell division requires FtsZ plus the essential Z ring-associated proteins FtsA and ZipA. Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP binding pocket and result in cell division arrest at non-permissive temperatures. We found that purified wild-type FtsA bound and hydrolysed ATP, whereas FtsA27 was defective in both activities. FtsA27 was also less able to localize to the Z ring in vivo. To investigate the role of ATP transactions in FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably, suppressors outside of the ATP binding site, including some mapping to the FtsA-FtsA subunit interface, also enhanced ATP transactions and exhibited gain of function phenotypes in vivo. These results suggest that allosteric effects, including changes in oligomeric state, may influence the ability of FtsA to bind and/or hydrolyse ATP.

Charge requirements of lipid II flippase activity in Escherichia coli.

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport.

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

A genetic screen for suppressors of a hypomorphic allele of Arabidopsis ARGONAUTE1.

ARGONAUTE1 (AGO1) encodes a key component of the complexes mediating microRNA (miRNA) function in Arabidopsis. To study the regulation, action and interactions of AGO1, we conducted a genetic screen to identify second-site mutations modifying the morphological phenotype of ago1-52, a partial loss-of-function allele of AGO1. Unlike null ago1 mutations, the hypomorphic ago1-52 allele does not cause lethality or sterility; however, ago1-52 does produce a morphological phenotype clearly distinct from wild type. In our screen for modifiers of ago1-52, we identified suppressor mutations that partially restore wild-type morphology in the ago1-52 background and we termed these mas (morphology of argonaute1-52 suppressed). We focused on 23 of these putative suppressors. Linkage analysis of the mas mutations together with sequencing of the AGO1 gene in genomic DNA and cDNA from ago1-52 mas plants indicated that 22 of the mas lines contain extragenic suppressors, and one contains an intragenic suppressor that affects splicing of ago1-52. In the presence of the wild-type allele of AGO1, most of the mas mutations cause a mild or no mutant phenotype on their own, indicating that the ago1-52 mutant may provide a sensitized background for examining the interactions of AGO1.

Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis.

Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.

Ethyl methanesulfonate induces mutations in Caenorhabditis elegans embryos at a high frequency

Mutagenesis protocols typically call for exposure of late-stage larvae or adults to a mutagen with the intention of inducing mutations in a robust germ line. Instead, ca. 16,000 CB665 [unc-58(e665)] one- to four-cell embryos of the nematode Caenorhabditis elegans were hand selected and exposed to ethyl methanesulfonate (EMS) for 50min. Twenty-one reversion mutants were recovered, of which 17 were intragenic suppressors of the e665 mutation. The mutation frequency was 6.5-fold higher than when CB665 adults were similarly mutagenized, which was predicted given that cell-cycle checkpoints are muted in C. elegans embryos. The mutation spectrum was similar to that obtained after standard EMS mutagenesis.

Three intragenic suppressors of a GTPase-deficient allele of GNAS associated with McCune-Albright syndrome.

Gain-of-function mutations in heterotrimeric G-protein α subunits are associated with a variety of human diseases. McCune-Albright syndrome (MAS) is caused by mutations in GNAS, the gene encoding Gs. Alterations at Arg201 significantly reduce the GTPase activity of the protein, rendering it constitutively active. In this study, we have constructed a library of random mutations in a constitutively active yeast GPA1 gene carrying a mutation homologous to the McCune-Albright allele (Arg297His). Intragenic suppressors found at sites with homology to the human Gs protein were tested for their ability to suppress the constitutive activity of an Arg201His mutation in Gs. Three intragenic suppressors, at Phe142, Arg231, and Leu266, were able to suppress elevated basal cAMP responses caused by Arg201His when expressed in HEK293 cells. A range of amino acid substitutions was introduced at each of these sites to investigate the chemical requirements for intragenic suppression. The ability of Gs proteins carrying the suppressor mutations alone to mediate receptor-induced cAMP production was measured. These results offer potential sites on Gs that could serve as drug targets for MAS therapies.

Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation.

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.

G673 could be a novel mutational hot spot for intragenic suppressors of pheS5 lesion in Escherichia coli

The pheS5 Ts mutant of Escherichia coli defined by a G293 → A293 transition, which is responsible for thermosensitive Phenylalanyl-tRNA synthetase has been well studied at both biochemical and molecular level but genetic analyses pertaining to suppressors of pheS5 were hard to come by. Here we have systematically analyzed a spectrum of Temperature-insensitive derivatives isolated from pheS5 Ts mutant and identified two intragenic suppressors affecting the same base pair coordinate G673 (pheS19 defines G673 → T673; Gly225 → Cys225 and pheS28 defines G673 → C673; Gly225 → Arg225). In fact in the third derivative, the intragenic suppressor originally named pheS43 (G673 → C673transversion) is virtually same as pheS28. In the fourth case, the very pheS5 lesion itself has got changed from A293 → T293 (named pheS40). Cloning of pheS+, pheS5, pheS5-pheS19, pheS5-pheS28 alleles into pBR322 and introduction of these clones into pheS5 mutant revealed that excess of double mutant protein is not at all good for the survival of cells at 42°C. These results clearly indicate a pivotal role for Gly225 in the structural/functional integrity of alpha subunit of E. coli PheRS enzyme and it is proposed that G673 might define a hot spot for intragenic suppressors of pheS5.

Characterizing intragenic suppressors of constitutively active alleles of Gs alpha (802.6)

Constitutive activity of Gs alpha is associated with several human diseases, including McCune‐Albright Syndrome (MAS). In this genetic disorder, Arg201 is mutated to His, Cys, or Ser, resulting in inhibition of GTP hydrolysis and subsequent overactivation of adenylyl cyclase and elevated basal cAMP levels. Previous studies identified three intragenic suppressor sites that blocked the constitutive activity caused by R201H: F142, R231, and L266. These mutations did not by themselves alter the activity of Gs alpha. The intragenic suppressors were examined for their ability to block the other MAS alleles. F142S and R231C suppressed the constitutive activity of both of MAS alleles. L266N suppressed R201S, and is currently being tested for the ability to block the constitutive activity of R201C. Thus, the intragenic suppressors may be useful drug targeting sites for MAS mutations generally, not just for R201H‐caused cases of the disease.Grant Funding Source: Supported by NIH grant 1R15ED020190‐01

Intragenic suppressors of McCune‐Albright syndrome R201H mutations also suppress other constitutively active Gs alleles (843.5)

McCune‐Albright Syndrome (MAS) is a genetic disorder caused by a mutation in Gs alpha at Arg201 that inhibits GTP hydrolysis, constitutively activating the protein. We identified three sites that, when mutated, suppressed the constitutive activity caused by R201H: F142, R231, and L266. None of the three mutations by themselves caused constitutive activity of the protein or significantly changed cellular responsiveness to hormone, as measured by the ability of a luteinizing hormone receptor agonist to activate cAMP production. We have now examined the ability of mutations at these three sites to suppress other activating mutations of Gs alpha. Other McCune‐Albright associated mutations include R201C and R201S. Both F142S and R231C were able to suppress the constitutive activity of both of previously untested R201 substitutions. L266N was able to suppress R201S, and experiments investigating its effects on R201C are ongoing. Gs alpha can also be constitutively activated by the Q227L substitution, associated with a variety of adenomas. The mutations identified as suppressors of R201H were all unable to suppress the constitutive activity of Q227L, suggesting that the action of the suppressors is specific to the R201 site and not generalizable to other activating perturbations of Gs alpha.Grant Funding Source: Supported by NIH grant 1R15ED020190‐01

TP53 Mutants in the Tower of Babel of Cancer Progression

Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, interfering, contact, structural, unfolded, misfolded, dimeric, monomeric, non-cooperative, unstable, supertrans, superstable, intragenic suppressor. TP53 mutants are many, more than 2,000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function (GoF); oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here, we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit GoF properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, which is beginning to impact cancer intervention strategies.

Arabidopsis HSP90 protein modulates RPP4‐mediated temperature‐dependent cell death and defense responses

Plant defense responses are regulated by temperature. In Arabidopsis, the chilling-sensitive mutant chs2-1 (rpp4-1d) contains a gain-of-function mutation in the TIR-NB-LRR (Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat) gene, RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4), which leads to constitutive activation of the defense response at low temperatures. Here, we identified and characterized two suppressors of rpp4-1d from a genetic screen, hsp90.2 and hsp90.3, which carry point mutations in the cytosolic heat shock proteins HSP90.2 and HSP90.3, respectively. The hsp90 mutants suppressed the chilling sensitivity of rpp4-1d, including seedling lethality, activation of the defense responses and cell death under chilling stress. The hsp90 mutants exhibited compromised RPM1 (RESISTANCE TO PSEUDOMONAS MACULICOLA 1)-, RPS4 (RESISTANCE TO P. SYRINGAE 4)- and RPP4-mediated pathogen resistance. The wild-type RPP4 and the mutated form rpp4 could interact with HSP90 to form a protein complex. Furthermore, RPP4 and rpp4 proteins accumulated in the cytoplasm and nucleus at normal temperatures, whereas the nuclear accumulation of the mutated rpp4 was decreased at low temperatures. Genetic analysis of the intragenic suppressors of rpp4-1d revealed the important functions of the NB-ARC and LRR domains of RPP4 in temperature-dependent defense signaling. In addition, the rpp4-1d-induced chilling sensitivity was largely independent of the WRKY70 or MOS (modifier of snc1) genes. [Correction added after online publication 11 March 2013: the expansions of TIR-NB-LRR and RPS4 were amended] This study reveals that Arabidopsis HSP90 regulates RPP4-mediated temperature-dependent cell death and defense responses.

Identification of Suppressors ofmbk-2/DYRKby Whole-Genome Sequencing

Screening for suppressor mutations is a powerful method to isolate genes that function in a common pathway or process. Because suppressor mutations often do not have phenotypes on their own, cloning of suppressor loci can be challenging. A method combining whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) mapping (WGS/SNP mapping) was developed to identify mutations with visible phenotypes in C. elegans. We show here that WGS/SNP mapping is an efficient method to map suppressor mutations without the need for previous phenotypic characterization. Using RNA-mediated interference to test candidate loci identified by WGS/SNP mapping, we identified 10 extragenic and six intragenic suppressors of mbk-2, a DYRK family kinase required for the transition from oocyte to zygote. Remarkably, seven suppressors are mutations in cell-cycle regulators that extend the timing of the oocyte-to-zygote transition.

Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

The Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV) that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W) residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S). The mutated OsARF(WS)s can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WS)s in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

Characterization of Two Second-Site Mutations Preventing Wild Type Protein Aggregation Caused by a Dominant Negative PMA1 Mutant

The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.