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E A Craig

Publications and source records attributed to E A Craig.

At least 55 records · Page 3Linked to original sources
PubMed1995

The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates.

hsp70 proteins of both eukaryotes and prokaryotes possess both ATPase and peptide binding activities. These two activities are crucial for the chaperone activity of hsp70 proteins. The activity of DnaK, the primary hsp70 of Escherichia coli, is modulated by the GrpE and DnaJ proteins. In the yeast Saccharomyces cerevisiae, the predominant cytosolic hsp70, Ssa1p, interacts with a DnaJ homologue, Ydj1p. In order to better understand the function of the Ssa1p/Ydj1p chaperone, the effects of polypeptide substrates and Ydj1p on Ssa1p ATPase activity were assessed using a combination of steady-state kinetic analysis and single turnover substrate hydrolysis experiments. Polypeptide substrates and Ydj1p both serve to stimulate ATPase activity of Ssa1p. The two types of effector are biochemically distinct, each conferring a characteristic K+ dependence on Ssa1p ATPase activity. However, in single turnover ATP hydrolysis experiments, both polypeptide substrates and Ydj1p destabilized the ATP.Ssa1p complex through a combination of accelerated hydrolysis of bound ATP and accelerated release of ATP from Ssa1p. The acceleration of ATP release by Ydj1p is a previously unidentified function of a DnaJ homologue. In the case of Ydj1p-stimulated Ssa1p, steady-state ATPase activity is increased less than 2-fold at physiological K+ concentrations, despite a 15-fold increase in the hydrolysis of bound ATP. The primary effect of Ydj1p appears to be to disfavor an ATP form of Ssa1p. On the other hand, peptide stimulation of Ssa1p ATPase activity was enhanced at physiological K+ concentrations, supporting the idea that cycles of ATP hydrolysis play an important role in the interaction of hsp70 with polypeptide substrates. The enhanced ATP dissociation caused by both polypeptide substrates and Ydj1p may play a role in the regulation of Ssa1p chaperone activity by altering the relative abundance of ATP-and ADP-bound forms.

Adenosine Diphosphate↗
PubMed1995

Mitochondrial import of subunit Va of cytochrome c oxidase characterized with yeast mutants.

We have investigated the unusual import pathway of cytochrome c oxidase subunit Va (COXVa) into the yeast mitochondrial inner membrane by use of mutants that lack import receptors or are defective in matrix hsp70. (i) Mitochondria lacking the receptor MOM72 are not impaired in import of COXVa. Mitochondria lacking the main receptor MOM19 are moderately reduced in import of COXVa; this, however, is caused by a reduction of the inner membrane potential and not by a lack of specific receptor functions. (ii) Mitochondria defective in the unfoldase function of matrix hsp70 efficiently import COXVa, whereas mitochondria defective in the translocase function of the hsp70 are blocked in import of COXVA. A COXVa construct where the internal hydrophobic sorting signal is placed close to the presequence does not require either hsp70 function. These results demonstrate that import of COXVa does not require MOM19 or MOM72, but they unexpectedly reveal a strong dependence on the translocase function of matrix hsp70. Two important implications about the characterization of mitochondrial protein import in general are obtained. First, the interpretation of import results with mutants lacking MOM19 have to consider effects on the membrane potential. Second, the distance between a matrix targeting sequence and a hydrophobic sorting sequence within a precursor appears to determine if the inner membrane sorting machinery can substitute for the translocase function of hsp70 or not.

Biological Transport↗
PubMed1995

Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins.

Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae. To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature. The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70. The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix. Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria. The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins. We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins.

Alleles↗
PubMed1995

A heat shock transcription factor with reduced activity suppresses a yeast HSP70 mutant.

Strains carrying deletions in both the SSA1 and SSA2 HSP70 genes of Saccharomyces cerevisiae exhibit pleiotropic phenotypes, including the inability to grow at 37 degrees C or higher, reduced growth rate at permissive temperatures, increased HSP gene expression, and constitutive thermotolerance. A screen for extragenic suppressors of the ssa1 ssa2 slow-growth phenotype identified a spontaneous dominant suppressor mutation, EXA3-1 (R.J. Nelson, M. Heschl, and E.A. Craig, Genetics 131:277-285, 1992). Here we report that EXA3-1 is an allele of HSF1, which encodes the heat shock transcription factor (HSF). Strains containing the EXA3-1 allele in a wild-type background exhibit a 10- to 15-fold reduction in HSF activity during steady-state growth conditions as well as a delay in the accumulation of the SSA4, HSP26, and HSP104 mRNAs after a heat shock. EXA3-1-mediated suppression is the result of a single amino acid substitution of a highly conserved residue in the HSF DNA-binding domain which drastically reduces the ability of HSF to bind to heat shock elements as evaluated by band shift analysis. Together, these results indicate that the poor growth of ssa1 ssa2 strains is the result, at least in part, of the overproduction of a deleterious heat shock protein(s). This conclusion is supported by the fact that the levels of at least some heat shock proteins are reduced in ssa1 ssa2 cells containing the EXA3-1 allele. Surprisingly, strains containing the EXA3-1 allele in a wild-type HSP70 background grow early as well as the wild-type strain over a wide temperature range, displaying only a slight reduction in growth rate at 37 degrees Celsius, indicating that cells contain significantly more HSF activity than is require for growth under steady-state conditions.

Base Sequence↗
PubMed1995

Posttranslational mitochondrial protein import in a homologous yeast in vitro system.

Posttranslational import of preproteins into mitochondria has been reported to be inefficient in a homologous yeast in vitro system, suggesting a requirement for coupling of protein synthesis and import. We have characterized a homologous yeast in vitro system which allows posttranslational mitochondrial import of preproteins. The efficiency is comparable to that of the heterologous system with rabbit reticulocyte lysate and isolated yeast mitochondria. Import in the homologous system depends on mitochondrial surface receptors, a membrane potential and the matrix heat shock protein Hsp70. Import is not blocked by the protein synthesis inhibitor cycloheximide, but is impaired by induction of stable folding in preproteins. Our studies demonstrate a posttranslational translocation mechanism in the homologous system, strongly supporting the validity of conclusions drawn from the widely used heterologous import system.

Animals↗
PubMed1995

Intercondylar shelf angle: a new method to determine race from the distal femur.

This study documents racial variation in the intercondylar shelf angle. The intercondylar shelf is actually the "roof" of the intercondylar notch, and on a lateral radiograph of the femur this is a distinctive feature called Blumensaat's Line. The angle between Blumensaat's Line and the posterior shaft of the femur can be quickly and easily measured. Variations in this angle are not dependent on the size of the femur, nor is the angle affected by arthritis in the notch or by trauma to the articular surfaces. Even fragmentary femora can be measured. This is a non-invasive technique that can be used in skeletal cases as well as cases where there are intact soft tissues.

Black People↗
PubMed1995

Nurse or doctor: biometry for intraocular lens power calculation, who should measure?

Biometry for intraocular lens power calculation is critical to the refractive success of cataract surgery with intraocular lens implantation. The majority of ophthalmology departments in Scotland currently entrust this procedure to junior medical staff. In an attempt to streamline preoperative assessment of cataract patients we have changed our practice so that nurses now carry out biometry. This prospective study, in which the results of 336 eyes undergoing cataract surgery and intraocular lens implantation were examined, shows that appropriately trained nursing staff in our department are able to perform the measurements for biometry at least as accurately as junior medical staff.

Bias↗
PubMed1995

The role of forensic anthropology in the recovery and analysis of Branch Davidian Compound victims: techniques of analysis.

The recovery and analysis of human remains from the Branch Davidian Compound, Mount Carmel, Texas, was a multidisciplinary team effort. This presentation deals with one aspect of the forensic anthropological contribution to this collaborative endeavor--the examination and inventory of human skeletal remains and the interpretation of these osteological data. Briefly described first are the content and format of the skeletal inventory and the examination procedures used for data collection. Two subsequent sections illustrate the use of these data to determine (a) age, sex, ancestry, stature, and other distinguishing characteristics, and (b) the presence and nature of any premortem, perimortem, and postmortem trauma. The next section shows how these findings assist in establishing positive identification through, for example, comparison with medical, dental, and other background records, especially comparison of premortem and postmortem radiographs of bones or dentition. In this context, we consider also the sorting of commingled remains, which includes preliminary assessment of rapidly decomposing remains prior to autopsy to preserve information crucial to identification.

Adolescent↗
PubMed1994

A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation.

The 70-kDa heat shock proteins (hsp70s) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate proteins coupled to cycles of ATP hydrolysis. In the prokaryote Escherichia coli, the hsp70 DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp70s and DnaJ-related proteins have been identified in eukaryotes, to our knowledge no GrpE-related proteins have been reported. We report the isolation and characterization of a eukaryotic grpE-related gene, MGE1. MGE1, an essential nuclear gene of the yeast Saccharomyces cerevisiae, encodes a soluble protein of the mitochondrial matrix. Cells with reduced expression of Mge1p accumulate the precursor form of a mitochondrial protein. Since mitochondrial hsp70 is required for translocation of precursors of mitochondrial proteins from the cytosol into the matrix of mitochondria, these data suggest that Mge1p acts in concert with mitochondrial hsp70 in protein translocation.

Amino Acid Sequence↗
PubMed1994

Heat-shock proteins as molecular chaperones.

Functional proteins within cells are normally present in their native, completely folded form. However, vital processes of protein biogenesis such as protein synthesis and translocation of proteins into intracellular compartments require the protein to exist temporarily in an unfolded or partially folded conformation. As a consequence, regions buried when a polypeptide is in its native conformation become exposed and interact with other proteins causing protein aggregation which is deleterious to the cell. To prevent aggregation as proteins become unfolded, heat-shock proteins protect these interactive surfaces by binding to them and facilitating the folding of unfolded or nascent polypeptides. In other instances the binding of heat-shock proteins to interactive surfaces of completely folded proteins is a crucial part of their regulation. As heat shock and other stress conditions cause cellular proteins to become partially unfolded, the ability of heat-shock proteins to protect cells against the adverse effects of stress becomes a logical extension of their normal function as molecular chaperones.

Animals↗
PubMed1994

Molecular evolution of the HSP70 multigene family.

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups.

Amino Acid Sequence↗
PubMed1994

Mitochondrial molecular chaperones: their role in protein translocation.

After synthesis in the cytosol, most mitochondrial proteins must traverse mitochondrial membranes to reach their functional location. During this process, proteins become unfolded and then refold to attain their native conformation after crossing the lipid bilayers. Mitochondrial molecular chaperones play an essential mechanistic role at various steps of this process. They facilitate presequence translocation, unfolding of the cytosol-localized domains of precursor proteins, movement across the mitochondrial membranes and, finally, folding of newly imported proteins within the matrix.

Biological Transport↗
PubMed1994

The protein import machinery of the mitochondrial inner membrane.

Mitochondria import most of their proteins from the cytosol. Although considerable information is available on the import machineries of the mitochondrial outer membrane and matrix, until recently little was known about the machinery of the inner membrane. Recent studies have identified three mitochondrial inner membrane proteins (MIMs) as essential components of the import machinery. MIM17 and MIM23 seem to form part of a channel, while MIM44, in cooperation with the heat-shock protein Hsp70, binds the preproteins in transit. The electrical membrane potential and ATP are needed to drive protein translocation through the MIM import machinery.

Adenosine Triphosphate↗
PubMed1994

Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA.

Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra-molecular complexes.

DNA, Mitochondrial↗
PubMed1994

Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44.

The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

Adenosine Triphosphate↗
PubMed1994

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.

Ascomycota↗