Adaptation of Candida albicans to growth on sorbose via monosomy of chromosome 5 accompanied by duplication of another chromosome carrying a gene responsible for sorbose utilization.

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C. albicans can evade commonly used antifungal agents are not fully understood. We are studying a model system involving growth of C. albicans on toxic sugar sorbose, which represses synthesis of cell wall glucan and, as a result, kills fungi in a manner similar to drugs from the echinocandins class. Adaptation to sorbose occurs predominantly due to reversible loss of one homolog of chromosome 5 (Ch5), which results in upregulation of the metabolic gene SOU1 (SOrbose Utilization) on Ch4. Here, we show that growth on sorbose due to Ch5 monosomy can involve a facultative trisomy of a hybrid Ch4/7 that serves to increase copy number of the SOU1 gene. This shows that control of expression of SOU1 can involve multiple mechanisms; in this case, negative regulation and increase in gene copy number operating simultaneously in cell.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.

Functional Subunits of Eukaryotic Chaperonin CCT/TRiC in Protein Folding.

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.

A synopsis of eukaryotic Nalpha-terminal acetyltransferases: nomenclature, subunits and substrates.

We have introduced a consistent nomenclature for the various subunits of the NatA-NatE N-terminal acetyltransferases from yeast, humans and other eukaryotes.

Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans.

N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-Delta nat1-Delta strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-Delta nat1-Delta strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.

Properties of Nat4, an N(alpha)-acetyltransferase of Saccharomyces cerevisiae that modifies N termini of histones H2A and H4.

Nat4, also designated NatD, was previously shown to acetylate the N termini of histones H2A and H4, which have SGGKG and SGRGK N termini (O. K. Song, X. Wang, J. H. Waterborg, and R. Sternglanz, J. Biol. Chem. 278:38109-38112, 2003). The analysis of chimeric proteins with various N-terminal segments of histone H4 fused to iso-1-cytochrome c revealed that efficient acetylation by NatD required at least 30 to 50 amino acid residues of the N terminus of histone H4. This requirement for an extended N terminus is in marked contrast with the major N-terminal acetyl transferases (NATs), i.e., NatA, NatB, and NatC, which require as few as two specific residues and usually no more than four or five. However, similar to the other NATs, NatD is associated with ribosomes. The nat4-Delta strain showed several minor phenotypes, including sensitivity to 3-aminotriazole, benomyl, and thiabendazole. Moreover, these nat4-Delta phenotypes were enhanced in the strain containing K5R K8R K12R replacements in the N-tail of histone H4, suggesting that the lack of N-terminal serine acetylation is synergistic to the lack of acetylation of the H4 N-tail lysines. Thus, N-terminal serine acetylation of histone H4 may be a part of an essential charge patch first described for the histone H2A.Z variant in Tetrahymena species.

Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.

Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes.

N-terminal acetylation is one of the most common modifications, occurring on the vast majority of eukaryotic proteins. Saccharomyces cerevisiae contains three major NATs, designated NatA, NatB, and NatC, with each having catalytic subunits Ard1p, Nat3p, and Mak3p, respectively. Gautschi et al. (Gautschi et al. [2003] Mol Cell Biol 23: 7403) previously demonstrated with peptide crosslinking experiments that NatA is bound to ribosomes. In our studies, biochemical fractionation in linear sucrose density gradients revealed that all of the NATs are associated with mono- and polyribosome fractions. However only a minor portion of Nat3p colocalized with the polyribosomes. Disruption of the polyribosomes did not cause dissociation of the NATs from ribosomal subparticles. The NAT auxiliary subunits, Nat1p and Mdm20p, apparently are required for efficient binding of the corresponding catalytic subunits to the ribosomes. Deletions of the genes corresponding to auxiliary subunits significantly diminish the protein levels of the catalytic subunits, especially Nat3p, while deletions of the catalytic subunits produced less effect on the stability of Nat1p and Mdm20p. Also two ribosomal proteins, Rpl25p and Rpl35p, were identified in a TAP-affinity purified NatA sample. Moreover, Ard1p copurifies with Rpl35p-TAP. We suggest that these two ribosomal proteins, which are in close proximity to the ribosomal exit tunnel, may play a role in NatA attachment to the ribosome.

Methylation of proteins involved in translation.

Methylation is one of the most common protein modifications. Many different prokaryotic and eukaryotic proteins are methylated, including proteins involved in translation, including ribosomal proteins (RPs) and translation factors (TFs). Positions of the methylated residues in six Escherichia coli RPs and two Saccharomyces cerevisiae RPs have been determined. At least two RPs, L3 and L12, are methylated in both organisms. Both prokaryotic and eukaryotic elongation TFs (EF1A) are methylated at lysine residues, while both release factors are methylated at glutamine residues. The enzymes catalysing methylation reactions, protein methyltransferases (MTases), generally use S-adenosylmethionine as the methyl donor to add one to three methyl groups that, in case of arginine, can be asymetrically positioned. The biological significance of RP and TF methylation is poorly understood, and deletions of the MTase genes usually do not cause major phenotypes. Apparently methylation modulates intra- or intermolecular interactions of the target proteins or affects their affinity for RNA, and, thus, influences various cell processes, including transcriptional regulation, RNA processing, ribosome assembly, translation accuracy, protein nuclear trafficking and metabolism, and cellular signalling. Differential methylation of specific RPs and TFs in a number of organisms at different physiological states indicates that this modification may play a regulatory role.

Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae.

We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Delta and rrp6-Delta. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this "retention signal" causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.

Phenotypes of yeast mutants lacking the mitochondrial protein Pet20p.

The pet20-delta deletion in Saccharomyces cerevisiae causes diminished growth on media containing non-fermentable carbon sources when incubated at both above and below the 30 degrees C optimal growth temperature. Furthermore, the pet20-delta strain has a greatly reduced level of cytochrome c, especially at 37 degrees C. The pet20-delta strain was sensitive to high NaCl and CaCl2 concentrations, hydrogen peroxide, oligomycin, polymixin B, amphotericin B and fluconazole. Biochemical fractionation and immunofluorescence staining demonstrated that Pet20p is located primarily in the mitochondria. Rhodamine B staining of pet20-delta cells showed an altered mitochondrial staining, indicating the possible lack of the mitochondrial membrane potential. We suggest that PET20 encodes a protein required for proper assembly or maintenance of mitochondrial components, but does not serve an enzymatic role. It is also possible that Pet20p may constitute a non-catalytic subunit of an uncharacterized mitochondrial complex or serve as a transporter or a coupling factor.

The yeast translation release factors Mrf1p and Sup45p (eRF1) are methylated, respectively, by the methyltransferases Mtq1p and Mtq2p.

The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N5-glutamine of the GGQ motif by PrmC methyltransferase. This motif is conserved in organisms from bacteria to higher eukaryotes. The Saccharomyces cerevisiae RFs, mitochondrial Mrf1p and cytoplasmic Sup45p (eRF1), have sequence similarities to the bacterial RFs, including the potential site of glutamine methylation in the GGQ motif. A computational analysis revealed two yeast proteins, Mtq1p and Mtq2p, that have strong sequence similarity to PrmC. Mass spectrometric analysis demonstrated that Mtq1p and Mtq2p methylate Mrf1p and Sup45p, respectively, in vivo. A tryptic peptide of Mrf1p, GGQHVNTTDSAVR, containing the GGQ motif was found to be approximately 50% methylated at the glutamine residue in the normal strain but completely unmodified in the peptide from mtq1-Delta. Moreover, Mtq1p methyltransferase activity was observed in an in vitro assay. In similar experiments, it was determined that Mtq2p methylates Sup45p. The Sup45p methylation by Mtq2p was recently confirmed independently (Heurgue-Hamard, V., Champ, S., Mora, L., Merkulova-Rainon, T., Kisselev, L. L., and Buckingham, R. H. (2005) J. Biol. Chem. 280, 2439-2445). Analysis of the deletion mutants showed that although mtq1-Delta had only moderate growth defects on nonfermentable carbon sources, the mtq2-Delta had multiple phenotypes, including cold sensitivity and sensitivity to translation fidelity antibiotics paromomycin and geneticin, to high salt and calcium concentrations, to polymyxin B, and to caffeine. Also, the mitochondrial mit(-) mutation, cox2-V25, containing a premature stop mutation, was suppressed by mtq1-Delta. Most interestingly, the mtq2-Delta was significantly more resistant to the anti-microtubule drugs thiabendazole and benomyl, suggesting that Mtq2p may also methylate certain microtubule-related proteins.

Candida albicans SOU1 encodes a sorbose reductase required for L-sorbose utilization.

Previous work in our laboratory showed that L-sorbose utilization in Candida albicans is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known and neither was the pathway for L-sorbose utilization. We have now expressed SOU1 at a high level from a replicative plasmid having a constitutive ADH1 promoter and purified a version of Sou1p tagged with the FLAG epitope at the N-terminus. Sou1FLAGNp has a sorbose reductase activity which utilizes NADPH as a co-factor and converts L-sorbose to D-sorbitol. It can also less efficiently utilize fructose as a substrate with NADPH as a co-factor, converting fructose to mannitol. In agreement with prediction, the purified enzyme has a subunit molecular weight of 31 kDa and a pI of about 4.8. It probably consists of four identical subunits and has a pH optimum of 6.2. The L-sorbose utilization pathway in C. albicans probably converts L-sorbose to fructose-6-phosphate via D-sorbitol as an intermediate. The first step is catalysed by Sou1p. We also found that C. albicans extracts have a D-sorbitol-6-phosphate dehydrogenase activity, not encoded by SOU1, which utilizes NADP as a co-factor. This activity has not been described previously in yeasts and may be involved in the conversion of phosphorylated D-sorbitol to fructose-6-phosphate or glucose-6-phosphate.

A nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae.

We previously demonstrated an increased degradation of mRNAs in mutants of Saccharomyces cerevisiae having blocks in nuclear export. The degradation activity, designated DRN (degradation of mRNA in the nucleus), requires Cbc1p, a nuclear cap-binding protein, and Rrp6p, a nuclear exosome component. Microarray procedures were used to determine the half-lives of mRNAs from normal and mutant strains, leading to the tentative identification of hundreds of normal mRNAs that were notably stabilized when either CBC1 or RRP6 were deleted. Northern blot analysis of representative mRNAs confirmed the diminished degradation. One representative of this group, SKS1 mRNA, was also shown by a cytological procedure to be preferentially retained in the nucleus compared with typical mRNAs. We suggest that all normal mRNAs are subjected to degradation by DRN, but the degree of degradation is determined by the degree of nuclear retention. Furthermore, these mRNAs particularly susceptible to DRN were also diminished by overproduction of Cbc1p, demonstrating a regulatory role for CBC1. This conclusion was corroborated by finding an inverse relationship of the CBC1 and SKS1 mRNA levels in normal strains grown under different conditions.

The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans.

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77K. The activities of complexes III and IV were determined in polarographic and enzyme-kinetic experiments using specific respiratory substrates and inhibitors. Results indicated that complexes III and IV are constitutively expressed and are functional in cells expressing AOX. Furthermore, the enzymatic activities of complexes III and IV were similar in mitochondrial preparations from cells grown with or without cyanide. We next investigated whether both pathways are simultaneously available for electron transfer from the Q pool to molecular oxygen. Respiration was virtually completely inhibited by the combination of cyanide and salicyl hydroxamic acid (SHAM) or antimycin A and SHAM, but only partly inhibited by either of these inhibitors alone. This indicates that electrons can in principle flow either through the conventional or the alternative respiratory pathway. The availability of two electron pathways in C. albicans and the potential use of either pathway endows this pleomorphic fungus with another level at which it can rapidly adjust to altered environmental conditions.

Cap-binding protein 1-mediated and eukaryotic translation initiation factor 4E-mediated pioneer rounds of translation in yeast.

Nonsense-mediated mRNA decay (NMD) in mammalian cells is restricted to newly synthesized mRNA that is bound at the 5' cap by the major nuclear cap-binding complex and at splicing-generated exon-exon junctions by exon junction complexes. This messenger ribonucleoprotein has been called the pioneer translation initiation complex and, accordingly, NMD occurs as a consequence of nonsense codon recognition during a pioneer round of translation. Here, we characterize the nature of messenger ribonucleoprotein that is targeted for NMD in Saccharomyces cerevisiae. Data indicate that NMD targets both cap-binding complex (Cbc)1p- and eukaryotic translation initiation factor (eIF)4E-bound mRNAs, unlike in mammalian cells, where NMD does not detectably target eIF4E-bound mRNA. First, intron-containing pre-mRNAs in yeast are detectably bound by either Cbc1p, or, unlike in mammalian cells, eIF4E, indicating that mRNAs can be derived from either Cbc1p- or eIF4E-bound pre-mRNAs. Second, the ratio of nonsense-containing Cbc1p-bound mRNA to nonsense-free Cbc1p-bound mRNA, which was < 0.4 for those mRNAs tested here, is essentially identical to the ratio of the corresponding nonsense-containing eIF4E-bound mRNA to nonsense-free eIF4E-bound mRNA, and both ratios increase in cells treated with the translational inhibitor cycloheximide (CHX). These data, together with data presented here and elsewhere showing that Cbc1p-bound transcripts are precursors to eIF4E-bound transcripts, demonstrate that Cbc1p-bound mRNA is targeted for NMD. In support of the idea that eIF4E-bound mRNA is also targeted for NMD, eIF4E-bound mRNA is targeted for NMD in strains that lack Cbc1p. These results suggest that both Cbc1p- and eIF4E-mediated pioneer rounds of translation occur in yeast.

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

The importance of mutation, then and now: studies with yeast cytochrome c.

The development of a genetic system based on the CYC1 gene was initiated over 40 years ago, primarily because of the anticipated ease of sequencing of the corresponding encoded protein, iso-1-cytochrome c from Saccharomyces cerevisiae. The success of the iso-cytochrome c system was dependent on the early development of methods for detecting and selecting cyc1 defective mutants and CYC1 functional revertants, and of methods for fine-structure genetic mapping using deletions and single-site mutations. The nonsense codons TAA and TAG, and the initiation codon ATG, were determined from the amino acid alterations of iso-1-cytochromes c from intragenic revertants; this represented the first assignments of such codons in a eukaryotic organism. The types of desired sequences were expanded by selecting recombinants from cyc1 x cyc1 nonfunctional mutants or CYC1 x CYC1 functional mutants, permitting the early determination of the rules of translation, which differed from those of prokaryotes by use of the most 5' AUG codon for initiation of translation. The sequence of 44 base pairs of CYC1 was determined with altered iso-1-cytochromes c from revertants of frameshift and initiation mutants, allowing the early cloning of the gene. A method was developed for transforming yeast directly with synthetic oligonucleotides, resulting in the convenient production of CYC1 mutants with defined sequences. At this point in time, Sherman and colleagues have published approximately 240 papers on or using the iso-cytochrome c system, dealing with such diverse topics as translation, informational suppressors, transcription and transcription termination, recombination, ectopic recombination, mutagen specificity, regulation by Ty1 elements, evolution of duplicated chromosomal segments, structure-function relationships of cytochrome c, protein stability and degradation, biosynthesis and mitochondrial import of cytochrome c, mitochondrial proteases, co- and post-translational modifications, and mRNA degradation. Current work on degradation of proteins in mitochondria, on degradation of mRNA in the nucleus, and on N-terminal acetylation stems from properties of CYC1 mutants isolated in early screens more than a decade ago.

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.