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F Sherman

Publications and source records attributed to F Sherman.

At least 73 records · Page 4Linked to original sources
PubMed1995

3'-end-forming signals of yeast mRNA.

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

Actins↗
PubMed1995

Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.

Amino Acid Sequence↗
PubMed1994

Thermodynamics of the equilibrium unfolding of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochromes c.

We report thermodynamic data for the chemical denaturation of iso-1-cytochromes c from Saccharomyces cerevisiae having amino acid substitutions R38A, N52I, and F82S in all possible combinations. The guanidine hydrochloride denaturation of isolated proteins was monitored by fluorescence measurements. The redox potentials, Eo', for both the folded and unfolded conformations have been measured. Free energy changes of chemical unfolding together with direct electrochemical measurement of the free energy changes of reduction for both the native and unfolded proteins yield a complete thermodynamic cycle, which includes four states of cytochrome c: oxidized folded, oxidized unfolded, reduced folded, and reduced unfolded. Completed cycles illustrate that the stability of cytochrome c to denaturing conditions is different for each amino acid substitution by an amount that depends on the heme oxidation state. Thus, the differential protein stability cannot be interpreted simply in terms of a hydrophobic effect, without also considering coupled Coulombic effects.

Base Sequence↗
PubMed1994

Differential scanning calorimetric study of the thermal unfolding transitions of yeast iso-1 and iso-2 cytochromes c and three composite isozymes.

The effects of regional sequence differences on the thermodynamic stability of a globular protein have been investigated by scanning microcalorimetry. Thermal transitions have been measured for two isozymes of yeast cytochrome c (iso-1-MS and iso-2) and three composite proteins (Comp1-MS, Comp2-MS, and Comp3-MS) in which amino acid segments are exchanged between the parental isozymes. There are three main observations. (1) In the temperature range of the unfolding transitions (40-60 degrees C) the unfolding free energies for the composite proteins are only slightly different from those of the parental isozymes, although in some cases there are large but compensating changes in the transitional enthalpy and entropy. At lower temperatures (0-30 degrees C), all the composites are significantly less stable than the two parental proteins. (2) Long-range structural effects are responsible for at least some of the observed differences in stability. For example, in the temperature range of the unfolding transitions (40-60 degrees C), the Comp1-MS protein which contains only a small amount of iso-2-like sequence is less stable than either of the parental isozymes, despite the fact that none of the iso-2-specific amino acid side chains impinges directly on any of the iso-1-specific amino acid side chains. (3) Changes in ionization of His 26 appear to be linked to thermal unfolding. Iso-1-MS and Comp1-MS contain a histidine residue at position 26 while iso-2 and the other two composites do not. On lowering the pH from pH 6 to 5, both iso-1-MS and Comp1-MS show a decrease in stability (lower Tm) within the unfolding transition region (40-60 degrees C), whereas the stabilities of iso-2, Comp2-MS, and Comp3-MS are essentially unchanged. The thermal unfolding transitions are highly reversible (> 95%) but mechanistically complex. A moderate dependence of Tm on protein concentration and the ratio of the van't Hoff enthalpy to the calorimetric enthalpy suggest that thermal unfolding involves the reversible association of a significant fraction of the unfolded species, at least at elevated protein concentrations.

Amino Acid Sequence↗
PubMed1994

Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast.

Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

Amino Acid Sequence↗
PubMed1994

NAT2, an essential gene encoding methionine N alpha-acetyltransferase in the yeast Saccharomyces cerevisiae.

N alpha-Acetylation is catalyzed by N alpha-acetyltransferases, which transfer acetyl groups from acetyl coenzyme A to the N termini of most eukaryotic proteins co-translationally. NAT1 and ARD1 from the yeast Saccharomyces cerevisiae (Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S., Gribskov, M., Colavito-Shepanski, M., Grunstein, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075) were previously shown to encode the major N alpha-acetyltransferase, which act on certain proteins having serine, glycine, and alanine but not methionine termini (Sherman, F., Moerschell, R. P., Tsunasawa, S., and Sternglanz, R. (1993) in Methods in Protein Sequence Analysis (Imahori, K., and Sakiyama, F., eds) pp. 173-181, Plenum Publishing Corp., New York). We have identified a second gene, NAT2, that may correspond to the N alpha-acetyltransferase acting on a subset of proteins having methionine termini. Crude extracts of a series of heat-sensitive mutants (Ts-) were screened for acetylation of a 24-amino acid synthetic peptide Met-Asn-Asn- in vitro. One mutant, nat2-1, out of 115 strains examined, lacked acetyltransferase activity, and the mutation co-segregated as a single gene with the heat-sensitive phenotype. The nat2-1 mutants were deficient in the ability to acetylate Met-Asn-Asn- and Met-Glu-Arg-peptides but were able to N alpha-acetylate Ser-Glu-Phe- and Ser-Tyr-Ser- peptides in vitro. The NAT2 wild-type gene was cloned by complementation of the nat2-1 mutant, and the DNA sequence revealed an open reading frame of 288 amino acids. Gene disruption demonstrated that NAT2 is an essential gene, and hybridization analysis indicated that it is located on chromosome VII. Furthermore, there was limited, but significant identities between the yeast N alpha-acetyltransferases Nat1, Ard1, Nat2, and Mak3, although no common motifs could be identified. We propose that NAT2 encodes the major N alpha-acetyl-transferase acting on certain proteins with only methionine termini, and that N alpha-acetylation of some of these proteins is essential for viability.

Acetylation↗
PubMed1994

The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c.

High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-1-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-1-cytochrome c the position and orientation of this water molecule 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.

Amino Acid Sequence↗
PubMed1994

PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae.

The PIM1 nuclear gene in the yeast Saccharomyces cerevisiae encodes a mitochondrial ATP-dependent protease that exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and one from Myxococcus xanthus. In addition, Pim1 is 1133 amino acids long and has a putative mitochondrial import signal in the N-terminal region. Enzymatic comparisons of normal PIM1+ and deficient pim1-delta strains revealed that the ATP-dependent protease is located within the mitochondrial matrix. The pim1-delta strains are unable to utilize nonfermentable substrates as the sole carbon source and are unable to maintain functional mitochondrial DNA, indicating that the Pim1 protease is required for mitochondrial function. PIM1 mRNA is constitutively expressed but is increased after thermal stress, suggesting that Pim1 may play a role in the heat shock response.

ATP-Dependent Proteases↗
PubMed1994

Physical constitution of ribosomal genes in common strains of Saccharomyces cerevisiae.

Several recent investigations, employing restriction endonucleases that do not cleave within rDNA units, revealed that a number of laboratory strains of Saccharomyces cerevisiae apparently contains a single tandem array of approximately 50 to 200 rDNA units on each chromosome XII homolog. The number of these rDNA units varies from strain to strain, among subclones of the same strain, and after different conditions of growth. In contrast, the commonly-used strain S288C and its derivatives contain two clusters on each chromosome XII homolog. Although the two clusters are stably maintained, the number of rDNA units within each cluster can vary as in strains with single clusters.

Chromosomes, Fungal↗
PubMed1994

Chromosomal alterations of Candida albicans are associated with the gain and loss of assimilating functions.

We have demonstrated that a normal laboratory strain of Candida albicans spontaneously produces mutants which acquire the ability to assimilate certain carbon sources that are not utilized by the parental strain. The examination of mutants acquiring the ability to utilize either sorbose or D-arabinose revealed a few additional phenotypic changes, including the gain and loss of the capacity to assimilate other carbon sources. The change of assimilation patterns resembled the polymorphic variation of assimilation patterns found among different wild-type strains of C. albicans. Most importantly, these sorbose- and D-arabinose-positive mutants were associated with chromosomal rearrangements, with each class of positive mutants having alterations of specific chromosomes. These findings demonstrated for the first time that chromosomal alterations in C. albicans are involved in genetic variation of fundamental functions of this asexual microorganism.

Arabinose↗
PubMed1993

The gene clusters ARC and COR on chromosomes 5 and 10, respectively, of Saccharomyces cerevisiae share a common ancestry.

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.

Amino Acid Sequence↗
PubMed1993

Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae.

Naturally occurring strains of Candida albicans are opportunistic pathogens that lack a sexual cycle and that are usually diploids with eight pairs of chromosomes. C. albicans spontaneously gives rise to a high frequency of colonial morphology mutants with altered electrophoretic karyotypes, involving one or more of their chromosomes. However, the most frequent changes involve chromosome VIII, which contains the genes coding for ribosomal DNA (rDNA) units. We have used restriction fragment lengths to analyze the number and physical array of the rDNA units on chromosome VIII in four normal clinical strains and seven morphological mutants derived spontaneously from one of the clinical isolates. HindIII does not cleave the rDNA repeats and liberates the tandem rDNA cluster from each homolog of chromosome VIII as a single fragment, whereas the cleavage at a single site by NotI reveals the size of the single rDNA unit. All clinical strains and morphological mutants differed greatly in the number of rDNA units per cluster and per cell. The four clinical isolates differed additionally among themselves by the size of the single rDNA unit. For a total of 25 chromosome VIII homologs in a total of 11 strains considered, the variability of chromosome VIII was exclusively due to the length of rDNA clusters (or the number of rDNA units) in approximately 92% of the cases, whereas the others involved other rearrangements of chromosome VIII. Only slight variations in the number of rDNA units were observed among 10 random C. albicans subclones and 10 random Saccharomyces cerevisiae subclones grown for a prolonged time at 22 degrees C. However, when grown faster at optimal temperatures of 37 and 30 degrees C, respectively, both fungi accumulated higher numbers of rDNA units, suggesting that this condition is selected for in rapidly growing cells. The morphological mutants, in comparison with the C. albicans subclones, contained a markedly wider distribution of the number of rDNA units, suggesting that a distinct process may be involved in altering the number of rDNA units in these mutants.

Candida albicans↗
PubMed1993

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.

Amino Acid Sequence↗
PubMed1993

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

Alleles↗
PubMed1992

NIP1, a gene required for nuclear transport in yeast.

Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.

Alleles↗
PubMed1992

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.

Amino Acid Sequence↗
PubMed1992

Effect of the Asn52----Ile mutation on the redox potential of yeast cytochrome c. Theory and experiment.

Theoretical methods for correlation of sequence changes and redox potential of electron transport proteins are examined using the Asn52----Ile mutation in cytochrome c as a test case. The first approach uses the protein dipoles Langevin dipoles (PDLD) method and the high resolution X-ray structures of the native and the mutant proteins. This approach is found to give reliable results where all the solvent molecules are represented by Langevin dipoles and also when some bound water molecules are represented explicitly. A free energy perturbation method is also found to give reasonable results but at the expense of much more computer time. Finally, an approach that generates mutant structures from the native structure by molecular dynamics simulation and then uses these configurations in PDLD calculations is found to give a reasonable estimate of the effect of the mutation on the corresponding redox potential. The encouraging results obtained here and in a preliminary test case of the Phe82----Ser mutation indicates that the present strategies can provide a useful tool for structure-redox and sequence-redox correlation in proteins.

Amino Acid Sequence↗