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The golf maintenance industry faces constant challenges to conserve water. Although golf courses have been pioneers in embracing highly efficient and smarter irrigation technologies, such as the use of recycled [...]

SHAM‐sensitive (STO) alternative respiration is present in the xylose‐metabolizing, Crabtree‐negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM‐sensitive terminal oxidase (PsSto1p), by genome walking from wild‐type CBS 6054 and subsequently deleted PsSTO1by targeted gene disruption. The resulting sto1‐Δ deletion mutant, FPL‐Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c1and a·a3did not change in the sto1‐Δ mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1‐Δ deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1in Saccharomyces cerevisiaeincreased its total oxygen consumption rate and imparted cyanide‐resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild‐type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non‐proton‐translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose‐metabolizing cells and that this non‐ATP‐generating route serves a regulatory function in the complex redox network of P. stipitis. Published in 2002 by John Wiley & Sons, Ltd.

Acetylation of proteins, either on various amino-terminal residues or on the ε-amino group of lysine residues, is catalyzed by a wide range of acetyltransferases. Amino-terminal acetylation occurs on the bulk of eukaryotic proteins and on regulatory peptides, whereas lysine acetylation occurs at different positions on a variety of proteins, including histones, transcription factors, nuclear import factors, and α-tubulin.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiaeis a mitochondrial protein; that the cyc2-Δ2deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome cdue to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Δ strains lacking the mitochondrial porin, Por1p, but not por2-Δ strains lacking the related porin, share some phenotypes similar to the cyc2-Δ2strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Δ2strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Δ2strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms.

A systematic screen for dominant-negative mutations of the CYT1gene, which encodes cytochromec1, revealed seven mutants after testing ∼104Saccharomyces cerevisiaestrains transformed with a library of mutagenized multicopy plasmids. DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G replacement, either by itself or accompanied with other amino acid replacements. A single R166G replacement produced by site-directed mutagenesis demonstrated that this change produced a nearly nonfunctional cytochromec1, with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c1having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromesc1and lies on the surface of Cyt1p in close proximity to the heme group but does not seem to interact directly with any of the physiological partners of the cytochromebc1complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochromec1but not for its assembly in the cytochromebc1complex.

The yeast Saccharomyces cerevisiaecontains three types of Nα-terminal acetyltransferases, NatA, NatB, and NatC, with each having a different catalytic subunit, Ard1p, Nat3p, and Mak3p, respectively, and each acetylating different sets of proteins with different Nα-terminal regions. We show that the NatC Nα-terminal acetyltransferases contains Mak10p and Mak31p subunits, in addition to Mak3p, and that all three subunits are associated with each other to form the active complex. Genetic deletion of any one of the three subunits results in identical abnormal phenotypes, including the lack of acetylation of a NatC substrate in vivo, diminished growth at 37 °C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.

Cytochromes cfrom plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome cof the yeast Saccharomyces cerevisiae. Testing of 6,144 strains of S. cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c. We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c. Unmethylated forms of cytochromecbut not other proteins or nucleic acids are methylatedin vitroby Ctm1p produced in S. cerevisiaeorEscherichia coli. Iso-1-cytochrome cpurified from a ctm1-Δ strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro. Like apocytochromec, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c. The ctm1-Δ strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome cwas produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome cwere coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.

Cytochrome creleased from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes cfrom higher eukaryotes can activate caspases in Xenopusegg and mammalian cytosols, iso-1 and iso-2 cytochromes cfrom the yeast Saccharomyces cerevisiaecannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72,N-ε-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome cexpressed in yeast. However, iso-1 cytochrome clacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes cthat lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

ABSTRACTThe cyc1-512mutation in Saccharomyces cerevisiaecauses a 90% reduction in the level of iso-1-cytochrome cbecause of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly longcyc1-512mRNAs which differ in length at their 3' termini. cyc1-512can be suppressed by deletion of either of the nonessential genes CBC1and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-? deletion suppressed the cyc1-512defect by diminishing degradation of the longer subset ofcyc1-512mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512defects by cbc1-? occurred by two different mechanisms. The levels of the shorter cyc1-512transcripts were enhanced in the cbc1-? mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longercyc1-512transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore,cbc1-? greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

Nα-Acetylation, catalyzed co-translationally with Nα-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants,nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the α1, α2, α3, α4, α7, and β3 subunits were acetylated with NAT1, the α5 and α6 subunits were acetylated with MAK3, and the β4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the α5 and α6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were Nα-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activitiesin vitro, suggesting thatNα-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1mutant than in the normal strain.

Although the gene responsible for Batten disease,CLN3,was positionally cloned in 1995, the function of Cln3p and the molecular basis of the disease still remain elusive. We previously reported that the yeastSaccharomyces cerevisiaecontains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance tod-(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol inbtn1-Δ yeast mutants. We have determined that yeast lacking Btn1p have an elevated ability to acidify media during growth that correlates with an elevated plasma membrane ATPase activity. Btn1p may be involved in maintaining pH homeostasis of yeast cells.

TheCLN3gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned theSaccharomyces cerevisiaehomolog to the humanCLN3gene, designatedBTN1,whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p,btn1-Δ deletion yeast strains, are more resistant tod-(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the humanCLN3gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced intoBTN1,altered the degree of ANP resistance. Severity of Batten disease due to mutations inCLN3and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.

The xylose‐utilizing yeast, Pichia stipitis,has a complex respiratory system that contains cytochrome and non‐cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome cgene (PsCYC1, Accession No. AF030426) was cloned from wild‐type P. stipitisCBS 6054 by cross‐hybridization to CYC1from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1showed 74% and 69% identity to ScCYC1and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1resulted in a mutant that uses the salicylhydroxamic acid (SHAM)‐sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes cand a·a3both disappeared in the cyc1‐Δ mutant, so no electron flow through the cytochrome coxidase was possible. The cyc1‐Δ mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1‐Δ mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0·46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0·38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen‐limiting conditions. Copyright © 1999 John Wiley & Sons, Ltd.

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)-sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a·a3 both disappeared in the cyc1-Δ mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Δ mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Δ mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0·46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0·38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen-limiting conditions. Copyright © 1999 John Wiley & Sons, Ltd.

A previous study demonstrated that Cyc2p from Saccharomyces cerevisiaeis a mitochondrial protein and that cyc2mutants contained only approximately 20% of the normal levels of cytochrome cdue to a partial deficiency in mitochondrial import of apo‐cytochrome c. We report herein that deletion of the entire gene results in defective mitochondrial function, as revealed by diminished growth on media containing non‐fermentable carbon sources. This defect is exacerbated in hyper‐ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a non‐ionic compound. We suggest that Cyc2p serves to maintain the osmotic stability of mitochondria, and its defect is exacerbated by KCl.

The Saccharomyces cerevisiaegene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c. Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast. © 1997 John Wiley & Sons, Ltd.

The yeast amber suppressor, SUP5-a, was previously shown to cause the insertion of tyrosine at the sites of UAG nonsense codons. Nucleotide sequencing established that this SUP5-asuppressor specifies a mutant tyrosine transfer RNA (tRNA) which has the anticodon CΨA instead of the normal GΨA, an alteration identical to that found in the tyrosine-inserting amber suppressor from Escherichia coli.

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-l-cytochrome cfrom the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-l-cytochrome cwas facilitated by the recently resolved three dimensional structure of iso-l-cytochrome c[(1987) J. Mol. Biol. 199, 295–314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-l-cytochrome cfunction appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-l-cytochrome c.

The yeast Saccharomyces cerevisiaecontains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genesCYC1and CYC7, respectively. The cytochromescare synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome cheme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome cbut not apo-iso-1-cytochrome cwas observed incyc3−mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome cwas due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochromecis dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochromecwas observed in pre2-2and pre1-1mutants having altered proteasome subunits; in ubc1,ubc4, and ubc5strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome cbut not apo-iso-2-cytochrome cis ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome cwas diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochromecis part of the regulatory process controlling the preferential expression of the iso-cytochromes c.

The CYC1 gene encoding cytochrome c in the yeast Candida albicans was cloned by complementation of a cytochrome c-deficient mutant of Saccharomyces cerevisiae, and its DNA sequence was determined. The analysis of the amino acid sequences of cytochrome c from 14 fungal species and two isoforms from S. cerevisiae revealed sequences unique to fungi, and revealed a phylogenetic relationship with a pronounced divergence between Schizosaccharomyces pombe and other ascomycetous budding yeast. The C. albicans CYC1 cytochrome c sequence has been assigned Accession Number U57896 in the GenBank/EMBL database. © 1997 John Wiley & Sons, Ltd.

The yeast Saccharomyces cerevisiae contains two clusters of eight genes each on chromosome 10 and 5, denoted, respectively, the COR and ARC regions. The genes in the COR region include TRS1 (a tRNA^Ser gene), ANB1, CYC1, UTR1, UTR3, OSM1, tRNA^Gly and RAD7 whereas the genes in the ARC region include TRS2 (a tRNA^Ser gene), TIF51A, UTR5, ANP1, RAD23, UTR4, CYC7 and UTR2. We have performed a physical analysis of the ARC region, including determining DNA sequence of the 7529 nucleotides; the open reading frames; the size and orientation of the transcripts; and the phenotypes resulting from deletions or gene disruptions. The ARC region was systematically compared to the COR region which was previously described. The gene pairs CYC1-CYC7 and ANB1-TIF51A were previously shown to be, respectively, approximately 80% and 90% identical. tRNA^Ser genes, TRS1 and TRS2, are located in both clusters 953 nt and 344 nt downstream of ANB1 and TIF51A, respectively. Some of the other gene pairs of these clusters are related in function and share only short segments of similarity distributed within the regions. The best alignment based on amino acid and nucleotide sequences indicates that the ARC and COR regions are ancestrally related by a duplication, a transposition, and a single rearrangement, followed by extensive divergence. These comparisons allowed an evaluation of distantly related sequences not obviously revealed by standard computer analysis. Surprisingly, the alignment suggested that a translated region of the ARC ANP1 gene and the COR tRNA^Gly gene are ancestrally related. Also translated regions of the COR gene RAD7 share similarities with both of the two adjacent ARC genes, ANP1 and RAD23. Five examples of simple repeated amino acid and DNA sequences occurred in the ARC region but none in the COR region. We suggest that these repeated sequences played a role in the divergence of ARC genes. Copyright 1993, 1999 Academic Press

Altered iso-1- and iso-2-cytochromes c, with certain amino acid replacements, occur at diminished levels due to degradation in the yeastSaccharomyces cerevisiae.A subclass of the labile isocytochromes c are significantly protected from degradation by the presence of cytochromes a·a3and c1, the physiological partners of cytochrome c. We have investigated the degradation that is dependent on physiological partners by examining the levels of iso-1-cytochrome c having all or most amino acid replacements at positions 6, 41, 52, and 78, in both ρ+strains and ρ−strains, which lacks cytochrome a·a3. In addition, we have examined some of these replacements in strains also having the N52I replacement, which suppresses a variety of abnormal iso-1-cytochromes c, including those whose degradation is either dependent or independent on the physiological partners. Although some degree of preferential ρ−-dependent reductions was observed for iso-1-cytochromes c having replacements at each of the 6, 41, 52, and 78 sites, prominent effects of ρ+/ρ−ratios of approximately 100/0 to 30/0 were observed for iso-1-cytochromes c having replacements mainly at the 41, 52, and 78 sites, but not the G6 site. We suggest that prominent degradation dependent on physiological partners may be restricted to certain regions of the cytochrome c molecule. Furthermore, we suggest that the region of the largest confirmational difference between oxidized and reduced cytochrome c appears to be particularly protected by interactions with its physiological partners.

Nip1p is an essential Saccharomyces cerevisiaeprotein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport. New results indicate that Nip1p has a primary role in translation initiation. Polysome profiles indicate that cells depleted of Nip1p and nip1-1cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells. Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits. Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni2+-NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p. The nip1-1ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46. Also, nip1-1mutant cells are hypersensitive to paromomycin. These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation.

High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-l-cytochrome c variants have been completed in both oxidation states. The most prominent structural difference observed in both mutant proteins is the displacement of a conserved, internally bound water molecule (Wat166) from the protein matrix. In wild-type yeast iso-l-cytochrome c the position and orientation of this water molecules is found to be dependent on the oxidation state of the heme iron atom. Overall our results suggest the function of Wat166 and its associated hydrogen bond network is threefold. First, the presence of Wat166 provides a convenient mechanism to modify the hydrogen bond network involving several residues near the Met80 ligand in an oxidation state dependent manner. Second, Wat166 is necessary for the maintenance of the spatial relationships between nearby side-chains and the hydrogen bond interactions formed between these groups in this region of the protein. An essential part of this role is ensuring the proper conformation of the side-chain of Tyr67 so that it forms a hydrogen bond interaction with the heme ligand Met80. This hydrogen bond influences the electron withdrawing power of the Met80 ligand and is therefore a factor in controlling the midpoint reduction potential of cytochrome c. Elimination of this interaction in the N52I-Y67F mutant protein or elimination of Wat166 in the N52I protein with the subsequent disruption in the position and interactions of the Tyr67 side-chain, leads to a drop of approximately 56 mV in the observed midpoint reduction potential of the heme group. Third. Wat166 also appears to mediate increases in the mobility of three nearby segments of polypeptide chain when cytochrome c is in the oxidized state. Previous studies have proposed these changes may be related to oxidation state dependent interactions between cytochrome c and its redox partners. Coincident with the absence of Wat166, such mobility changes are not observed in the N52I and N52I-Y67F mutant proteins. It is possible that much of the increased protein stability observed for both mutant proteins may be due to this factor. Finally, our results show that neither heme iron charge nor heme plane distortion are responsible for oxidation state dependent conformational changes in the pyrrole A propionate region. Instead, the changes observed appear to be driven by the change in conformation that the side-chain of Asn52 experiences as the result of oxidation state dependent movement of Wat166. Copyright 1994, 1999 Academic Press

The Ψ+extrachromosomal determinant in the yeast Saccharomyces cerevisiaesuppresses certain UAA markers and increases the efficiency of suppression of UAA suppressors and certain frameshift suppressors. Although the exact nature of Ψ+determinant is unknown, it is believed to be a self-replicating cytoplasmic factor affecting some component of the translational machinery. In this report we describe growth conditions for efficient mutation or elimination of the Ψ+determinant. Incubation of Ψ+cultures in hypertonic nutrient medium resulted in rapid conversion to a culture containing predominantly Ψ-cells during the growth cycle. The kinetics of Ψ+to Ψ-conversion established that the occurrence of Ψ-cells was due to induction and not to selection of pre-existing Ψ-cells. The results suggest that the replication of the Ψ+determinant is sensitive to hypertonic conditions.

Altered peptides from chymotryptic digests of iso-1-cytochromes cthat were obtained from two revertants of the cyc1–6 mutant, one revertant of cyc1–10 mutant, and one revertant of the cyc1–15 mutant have been isolated and their amino acid sequences established. The single amino acid replacements, serine and threonine at position 12 in two of the revertant iso-1-cytochromes c, indicated that cyc1–6 is a missense mutant which contains a residue of proline at position 12 instead of the normal residue of alanine. The RNA codons for proline differ from the RNA codons of alanine, serine, and threonine by one base, indicating that the cyc1–6 mutant arose by a G·C → C·G transversion and the serine and threonine revertants were formed, respectively, by a G·C → A·T transition and a G·C → T·A transversion. The revertant from cyc1–10 contained a residue of asparagine at position 13 in place of the normal residue of threonine, and the revertant from cyc1–15 contained a residue of isoleucine at position 17 in place of the normal residue of threonine. While it has not been possible to deduce the exact nature of the mutational changes in the cyc1–10 and cyc1–15 mutants since only one altered iso-1-cytochrome cwas characterized from each of these cyc1 mutants, it is clear that these replacements are not compatible with mutations by way of any of the known nonsense codons. The complete deficiency of iso-1-cytochrome cin the cyc1–10 mutant and the amino acid replacement in the revertant could be explained either by frameshift mutations, if the forward and reverse mutations occurred within a single codon, or more likely by a missense mutation that gave rise to a lysine residue at this site which is incompatible for as yet undefined steps in the attachment of the heme group. We have no simple explanation for the cyc1–15 mutation since the site of the amino acid replacement in the revertant is not compatible with the site of the mutation that was determined by genetic fine structure mapping.

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.

We have identified a gene in maize that encodes a product belonging to the Lon protease family. In yeast and mammals, Lon-type proteases catalyze the ATP-dependent degradation of mitochondrial matrix proteins. The maize gene, which we have designated LON1, is predicted to encode a protein with a molecular mass of 97.7 kDa. Lon1p is more similar in sequence to bacterial Lon proteases than to the yeast and human mitochondrial Lon proteases. LON1 transcripts are present in shoots of 4-day-old etiolated maize seedlings, and transcript levels decrease when these seedlings are heat-shocked. LON1 transcripts are also present at comparable levels in leaves and roots of 2-week-old greenhouse-grown seedlings. In yeast, the mitochondrial Lon-type protease, Pim1p, has been implicated in mitochondrial protein turnover, the assembly of mitochondrial enzyme complexes, and mitochondrial DNA maintenance, and it is essential for respiratory function. We show that maize Lon1p can replace the Pim1p function in yeast for maintaining mitochondrial DNA integrity, but not in the assembly of cytochrome a _ a3 complexes.

Summary This study dealt with the ability of non-denaturing gel electrophoresis to separate iso-1-cytochromec with single amino acid replacements isolated from revertants of variouscyc1 nonsense mutants of the yeastSaccharomyces cerevisiae. A total of 28 different iso-1-cytochromesc with single amino acid substitutions of one of seven amino acids at six positions were examined on nondenaturing polyacrylamide gels at pH 4.8. Each of these iso-1-cytochromesc exhibited 1 of 16 distinct electrophoretic mobilities. We could distinguish the majority of iso-1-cytochromesc, even those having the same replacement at different sites and those having different replacements that resulted in the same net charge. These results provide confirmation of the importance of site-specific effects on the electrophoretic mobility, and presumably other properties, of proteins differing in sequence by as little as one amino acid. They demonstrate that nondenaturing electrophoresis is able to separate the majority of, but not all, proteins differing by single amino acids.

The Saccharomyces cerevisiaetemperature-sensitive (ts) allele nip7-1exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+gene complemented the nip7-1ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

Approximately one-half of the mutants of Saccharomyces cerevisiaethat are selected as resistant to methyl mercury are also found to require methionine. Eighty-four percent of these metmutations occur at the met15 locus, and the remaining 16% occur at the met2 locus. Surprisingly, the methionine-requiring mutants are recovered at a much higher frequency on methionineless media than on media supplemented with methionine. Growth patterns of the metmutants on media having a continuous concentration gradient of methionine and mercury compounds indicate that, at a critical concentration of the mercury compounds, the methionine requirement of certain metmutants is partially or completely alleviated. This was found for met2, met15, and to a lesser extent for met6, but not for any other methionine mutants. This loss of methionine requirement is produced with methyl mercury, phenyl mercury, and mercuric chloride although met2 and met15 strains can be shown to be resistant only to methyl mercury. Other methionine auxotrophs are not resistant to any of the three mercury compounds. The met2 and met15 mutants, but not the other methionine auxotrophs, develop a sheen of an unidentified product when grown on media with mercuric chloride but not with methyl mercury or phenyl mercury. It is suggested that met2 and met15 mutants produce a simple diffusible substance, which detoxifies methyl mercury, which reacts with mercuric chloride to produce a sheen, and which is the cause of the methionine requirement.

We have confirmed by spectral analysis that cytochrome oxidase is not present in strains of the yeast Saccharomyces cerevisiaehaving a primary deficiency in cytochrome c, and we have demonstrated by immunological procedures that such strains lack the mitochondrially encoded subunits I, II, and III of cytochrome oxidase. Furthermore, pulse-chase experiments demonstrated that subunit II is rapidly degraded in vivo. This degradation can be at least partially suppressed by disruption of the nuclear gene YME1, which encodes a putative ATP-Zn2+-dependent protease. We suggest that the cytochrome oxidase subunits are not properly assembled in the absence of cytochrome c, and that Yme1 and possibly other proteases degrade the unassembled mitochondrial-encoded subunits of cytochrome oxidase.

IT has been known for several years that strains resistant to mercury can be obtained in several bacterial species1–4. Soon after the correlation between resistance to antibiotics and to mercury was recognised2,5, it was established that genetic elements conferring resistance to antibiotics, mercury and other heavy metals in Escherichia coli and Salmonella typhimurium6and Staphylococcus aureus7,8reside on extrachromosomal resistance transfer factors or plasmids. Among fungi, mercury resistant strains of Botrytis cinerea9, Penicillium notatum, Sclerotinia fructicola, Stemphylium sarcinaeforme10, and Saccharomyces cerevisiae11,12have been reported. In most cases1,3,9–12, this was accomplished by ‘training’ the normal strains for growth on media supplemented with successively increasing concentrations of mercury compounds, and in some cases10the resistance was lost when subcultured on mercury-free media. It is noteworthy that in none of the mercury-adapted strains of fungi has the genetic basis of resistance been determined. In this report we describe a method of isolation and characterisation of methyl mercury resistant mutants of S. cerevisiae. This study was undertaken with the view that the examination of physiological changes associated with genetically defined resistant mutants will be useful in studying the mechanisms of cellular detoxification of organic mercurials.

STREPTOMYCIN, an aminoglycoside antibiotic, can reverse the mutant phenotypes of many nonsense and missense mutations in Escherichia coliand in bacteriophage T4. This phenomenon has been called phenotypic suppression, since the mutant phenotype returns after removal of the drug1. The most likely explanation for phenotypic suppression is that streptomycin promotes mistranslation in vivo, and that acceptable amino acids are inserted into the growing polypeptide chain at the site of the mutant codon. Consistent with this view is the observation that streptomycin causes E. coliribosomes to mistranslate RNA in vitro2,3. Streptomycin and neomycin have however been found to have no effect in stimulating ribosomes from eukaryotic cells to mistranslate RNA in vitro4,5. A subclass of the aminoglycoside antibiotics has been shown6,7to stimulate eukaryotic ribosomes to misread RNA. The highly active molecules are distinguished in that they contain the drug fragment paromamine (or 3′-deoxyparomamine). We have therefore examined the capacity of various aminoglycosides to suppress mutations phenotypically in the eukaryotic yeast, Saccharomyces cerevisiae. The results presented here show that paromomycin, which contains paromamine, is capable of phenotypic suppression of the nonsense mutations in S. cerevisiae.

We have examined the tRNAs of two related strains of Saccharomyces cerevisiae, ?+ and ?-, which differ with respect to an extrachromosomal genetic element that modulates the expression of genotypic and phenotypic suppression. Both the pattern of tRNAs synthesized and the level of nucleotide modification of several selected tRNA species were found to be the same in the ?+ and ?- strains.

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Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as shown for GroEL. Unlike GroEL, CCT does not have GroES-like cofactor, rather it has a built-in lid structure responsible for closing the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover, prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates which are linked to multiple pathological disorders.

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