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Biomedical subjects

F Sherman

Publications and source records attributed to F Sherman.

At least 19 recordsLinked to original sources
PubMed2001

Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due 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-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, 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-Delta2 strain. 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.

Adaptor Proteins, Signal Transducing↗
PubMed2001

NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p.

The yeast Saccharomyces cerevisiae contains three types of N(alpha)-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 N(alpha)-terminal regions. We show that the NatC N(alpha)-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 degrees C on media containing nonfermentable carbon sources, and the lack of maintenance or assembly of the L-A dsRNA viral particle.

Amino Acid Sequence↗
PubMed2001

Role of Arg-166 in yeast cytochrome C1.

A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c(1), revealed seven mutants after testing approximately 10(4) Saccharomyces cerevisiae strains 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 cytochrome c(1), with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c(1) having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromes c(1) and 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 cytochrome bc(1) complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochrome c(1) but not for its assembly in the cytochrome bc(1) complex.

Amino Acid Sequence↗
PubMed2001

UV and X-ray sensitivity of Candida albicans laboratory strains and mutants having chromosomal alterations.

Utilization of L-sorbose, D-arabinose or primary fluconazole resistance in Candida albicans are controlled by copy number of specific chromosomes. On the other hand, spontaneous morphological mutants have a wide range of chromosomal alterations. We have investigated the UV and X-ray sensitivity of these mutants, as well as C. albicans laboratory strains. While L-sorbose utilizing mutants had normal sensitivities, a large subclass of D-arabinose utilizing mutants was abnormally sensitive to UV. Spontaneous morphological mutants responded differently, an expected result because of the heterogeneous nature of their electrophoretic karyotypes. We suggest that the differences in UV and X-ray sensitivity are due to gene imbalance caused by some chromosomal alterations. In this respect, the radiation sensitivity is similar to other features impaired by changes in chromosomes, but is unlike the acquisition of the ability to utilize alternative nutrients or the acquisition of resistance to fluconazole. Our studies also revealed that strains of C. albicans heterozygous for the mating type loci exhibited the same X-ray sensitivity as homozygous or hemizygous strains, a finding which is in contrast to the properties of Saccharomyces cerevisiae, where heterozygous strains are more resistant. This feature of C. albicans strains may be indicative of an inefficient repair system that may be related to inefficiency of mating.

Journal Article↗
PubMed2000

Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.

Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.

Amino Acid Sequence↗
PubMed2000

Cytochrome c methyltransferase, Ctm1p, of yeast.

Cytochromes c from 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 c of 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 cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S. cerevisiae or Escherichia coli. Iso-1-cytochrome c purified from a ctm1-Delta 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 apocytochrome c, 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-Delta strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.

Amino Acid Sequence↗
PubMed2000

Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation.

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking 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 c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

Amino Acid Sequence↗
PubMed2000

N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome.

N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-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 alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-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 nat1 mutant than in the normal strain.

Acetylation↗
PubMed2000

The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis.

The BTN1 gene product of the yeast Saccharomyces cerevisiae is 39% identical and 59% similar to human CLN3, which is associated with the neurodegenerative disorder Batten disease. Furthermore, btn1-Delta strains have an elevated activity of the plasma membrane H(+)-ATPase due to an abnormally high vacuolar acidity during the early phase of growth. Previously, DNA microarray analysis revealed that btn1-Delta strains compensate for the altered plasma membrane H(+)-ATPase activity and vacuolar pH by elevating the expression of the two genes HSP30 and BTN2. We now show that deletion of either HSP30 or BTN2 in either BTN1(+) or btn1-Delta strains does not alter vacuolar pH but does lead to an increased activity of the vacuolar H(+)-ATPase. Deletion of BTN1, BTN2, or HSP30 does not alter cytosolic pH but diminishes pH buffering capacity and causes poor growth at low pH in a medium containing sorbic acid, a condition known to result in disturbed intracellular pH homeostasis. Btn2p was localized to the cytosol, suggesting a role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase. Increased expression of HSP30 and BTN2 in btn1-Delta strains and diminished growth of btn1-Delta, hsp30-Delta, and btn2-Delta strains at low pH reinforce our view that altered pH homeostasis is the underlying cause of Batten disease.

Culture Media↗
PubMed2000

The role of nuclear cap binding protein Cbc1p of yeast in mRNA termination and degradation.

The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and 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-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, 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 cyc1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

Fungal Proteins↗
PubMed1999

The action of N-terminal acetyltransferases on yeast ribosomal proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the state of N-terminal acetylation of 68 ribosomal proteins from a normal strain of Saccharomyces cerevisiae and from the ard1-Delta, nat3-Delta, and mak3-Delta mutants (), each lacking a catalytic subunit of three different N-terminal acetyltransferases. A total 30 of the of 68 ribosomal proteins were N-terminal-acetylated, and 24 of these (80%) were NatA substrates, unacetylated in solely the ard1-Delta mutant and having mainly Ac-Ser- termini and a few with Ac-Ala- or Ac-Thr- termini. Only 4 (13%) were NatB substrates, unacetylated in solely the nat3-Delta mutant, and having Ac-Met-Asp- or Ac-Met-Glu- termini. No NatC substrates were uncovered, e.g. unacetylated in solely mak3-Delta mutants, consistent with finding that none of the ribosomal proteins had Ac-Met-Ile-, Ac-Met-Leu-, or Ac-Met-Phe- termini. Interestingly, two new types of the unusual NatD substrates were uncovered, having either Ac-Ser-Asp-Phe- or Ac-Ser-Asp-Ala- termini that were unacetylated in the ard1-Delta mutant, and only partially acetylated in the mak3-Delta mutant and, for one case, also only partially in the nat3-Delta mutant. We suggest that the acetylation of NatD substrates requires not only Ard1p and Nat1p, but also auxiliary factors that are acetylated by the Mak3p and Nat3p N-terminal acetyltransferases.

Acetylation↗
PubMed1999

Water determination in drugs containing thiols.

A new rapid analytical method is applied for water determination in alpha-Mono-thioglycerol and Captopril tablets containing thiols, and therefore, not amenable for direct Karl Fischer titration. The method is based on the consecutive titration first of thiol by a novel reagent, and then of water by a conventional K. Fischer reagent in the same sample and cell with the electrometric 'dead-stop' location of the end point in both titrations. The new reagent consists of iodine, potassium iodide and sodium acetate in non-aqueous medium. Estimated repeatability and accuracy of both water and thiol determinations are satisfactory.

Captopril↗
PubMed1999

Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation.

Acetylation↗
PubMed1999

Phenotypic reversal of the btn1 defects in yeast by chloroquine: a yeast model for Batten disease.

BTN1 of Saccharomyces cerevisiae encodes an ortholog of CLN3, the human Batten disease gene. We have reported previously that deletion of BTN1, btn1-Delta, resulted in a pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP). This phenotype was caused by btn1-Delta strains having an elevated ability to acidify growth medium through an elevated activity of the plasma membrane H(+)-ATPase, resulting from a decreased vacuolar pH during early growth. We have determined that growing btn1-Delta strains in the presence of chloroquine reverses the resistance to ANP, decreases the rate of medium acidification, decreases the activity of plasma membrane H(+)-ATPase, and elevates vacuolar pH. However, an additional effect of this phenotypic reversal is that activity of plasma membrane H(+)-ATPase is decreased further and vacuolar pH is increased further as btn1-Delta strains continue to grow. This phenotypic reversal of btn1-Delta can be considered for developing a therapy for Batten disease.

Chloroquine↗
PubMed1999

Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production.

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.a(3) both disappeared in the cyc1-Delta mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Delta mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Delta 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.

Antifungal Agents↗
PubMed1999

Studies of pH regulation by Btn1p, the yeast homolog of human Cln3p.

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 yeast Saccharomyces cerevisiae contains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1, 3-propanediol in btn1-Delta 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.

Cell Membrane↗
PubMed1999

Investigation of Batten disease with the yeast Saccharomyces cerevisiae.

The CLN3 gene, 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 the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and 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.

Cell Division↗