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E A Craig

Publications and source records attributed to E A Craig.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae.

Iron is fundamental to many biological processes, but is also detrimental as it fosters the synthesis of destructive oxygen radicals. Recent experiments have increased our knowledge of the critical process of regulation of mitochondrial iron metabolism. A number of genes directly involved in iron homeostasis in this organelle have been identified. Intriguingly, a minor Hsp70 molecular chaperone of the mitochondrial matrix has been implicated as a player in this process as well.

HSP70 Heat-Shock Proteins↗
PubMed1998

The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains.

The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of Saccharomyces cerevisiae are functionally distinct. Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties. The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70. Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity. In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K+ whereas Ssb activity does not. Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a Km and Vmax for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb. Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb. Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself. The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.

Adenosine Triphosphatases↗
PubMed1998

Folding in vivo of a newly translated yeast cytosolic enzyme is mediated by the SSA class of cytosolic yeast Hsp70 proteins.

The nature of chaperone action in the eukaryotic cytosol that assists newly translated cytosolic proteins to reach the native state has remained poorly defined. Actin, tubulin, and Galpha transducin are assisted by the cytosolic chaperonin, CCT, but many other proteins, for example, ornithine transcarbamoylase (OTC), a cytosolic homotrimeric enzyme of yeast, do not require CCT action. Here, we observe that yeast cytosolic OTC is assisted to its native state by the SSA class of yeast cytosolic Hsp70 proteins. In vitro, refolding of OTC diluted from denaturant was assisted by crude yeast cytosol and ATP and found to be directed by SSA1/2. In vivo, when OTC was induced in a temperature-sensitive SSA-deficient strain, it exhibited reduced specific activity, and nonnative subunits were detected in the soluble fraction. These findings indicate that, in vivo, the Hsp70 system assists in folding at least some newly translated cytosolic enzymes, most likely functioning in a posttranslational manner.

Adenosine Triphosphate↗
PubMed1998

Zuotin, a ribosome-associated DnaJ molecular chaperone.

Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins. In Saccharomyces cerevisiae, SSB proteins are ribosome-associated Hsp70s which interact with the newly synthesized nascent polypeptide chain. Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ-related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity. Zuo1, which has been shown previously to be a nucleic acid-binding protein, is also a ribosome-associated protein localized predominantly in the cytosol. Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA. We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome.

Cytosol↗
PubMed1998

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex.

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Adenosine Triphosphate↗
PubMed1998

Isolation of UBP3, encoding a de-ubiquitinating enzyme, as a multicopy suppressor of a heat-shock mutant strain of S. cerevisiae.

Yeast strains lacking functional copies of the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, are temperature-sensitive. In this report, we describe the isolation of a high-copy suppressor of this temperature sensitivity, UBP3, which encodes a de-ubiquitinating enzyme. We show that ubp3 mutant yeast strains have a mild slow-growth phenotype and accumulate ubiquitin-protein conjugates. We propose a model in which Ubp3p acts in vivo to reverse the ubiquitination of substrate proteins, allowing temporarily misfolded proteins an opportunity to fold correctly.

Adenosine Triphosphatases↗
PubMed1998

Suppression of an Hsp70 mutant phenotype in Saccharomyces cerevisiae through loss of function of the chromatin component Sin1p/Spt2p.

The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast Saccharomyces cerevisiae has four members, encoded by SSA1, SSA2, SSA3, and SSA4. Deletion of the two constitutively expressed genes, SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of ssa1 ssa2 strains, EXA1-1, is a loss-of-function mutation in SIN1/SPT2, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression of SSA3 in the ssa1 ssa2 mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for SSA genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of SSA3 in the ssa1 ssa2 mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of SSA3 which is modulated by Sin1p. The EXA1-1 suppressor mutation thus improves the growth of ssa1 ssa2 strains by selectively increasing HSF-mediated expression of SSA3.

Bacterial Proteins↗
PubMed1997

A mutant form of mitochondrial GrpE suppresses the sorting defect caused by an alteration in the presequence of cytochrome b2.

Transport of preproteins across the inner mitochondrial membrane requires the action of the matrix heat shock protein Hsp70. Together with its co-chaperone mitochondrial GrpE (Mge1), mtHsp70 transiently binds to the inner membrane translocase subunit Tim44 in a nucleotide-regulated manner, forming an ATP-dependent import driving machinery. We report that a mutant form of Mge1 (Mge1-100) is completely absent in mtHsp70-Tim44 complexes, although its ability to interact with soluble mtHsp70 is only partially reduced. While this mge1-100 mutation only partially retards preprotein translocation into the matrix, it exerts a selective effect on sorting of cytochrome b2 to the intermembrane space. A cytochrome b2 with an altered sorting signal, which is only processed to the intermediate stage and mistargeted to the matrix of wild-type mitochondria, is processed to the mature form and correctly targeted to the intermembrane space of mge1-100 mitochondria. These results suggest that (1) Mge1-100 discriminates between soluble and membrane-bound mtHsp70 and (2) the membrane-bound mtHsp70-Mge1 driving system competes with the sorting machinery for translocation of preproteins like cytochrome b2.

Blotting, Western↗
PubMed1997

Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae.

Mge1, a GrpE-related protein in the mitochondrial matrix of the budding yeast Saccharomyces cerevisiae, is required for translocation of precursor proteins into mitochondria. The effect of Mge1 on nucleotide release from Ssc1, an Hsp70 of the mitochondrial matrix, was analyzed. The release of both ATP and ADP from Ssc1 was stimulated in the presence of Mge1, therefore we conclude that Mge1 functions as a nucleotide release factor for Ssc1. Mge1 bound stably to Ssc1 in vitro; this interaction was resistant to high concentrations of salt but was disrupted by the addition of ATP. ADP was much less effective in releasing Mge1 from Ssc1 whereas ATP gamma S and AMPPNP could not disrupt the Ssc1/Mge1 complex. Ssc1-3, a temperature sensitive SSC1 mutant protein, did not form a detectable complex with Mge1. Consistent with the lack of a detectable interaction, Mge1 did not stimulate nucleotide release from Ssc1-3. A conserved loop structure on the surface of the ATPase domain of DnaK has been implicated in its interaction with GrpE. Since the single amino acid change in Ssc1-3 lies very close to the analogous loop in Ssc1, the role of this loop in the Ssc1:Mge1 interaction was investigated. Deletion of the loop abolished the physical and functional interaction of Ssc1 with Mge1, suggesting that the loop in Ssc1 is also important for the Ssc1:Mge1 interaction. Two mutants with single amino acid changes within the loop did not eliminate the stable binding of Mge1, yet the binding of Mge1 did not stimulate the release of nucleotides from the mutant SSC1 proteins. We propose that the loop region of Ssc1 is important for the physical interaction between Mge1 and Ssc1, and for generation of a conformational change necessary for Mge1-induced nucleotide release.

Adenine Nucleotides↗
PubMed1997

Functional specificity among Hsp70 molecular chaperones.

Molecular chaperones of the 70-kilodalton heat shock protein (Hsp70) class bind to partially unfolded polypeptide substrates and participate in a wide variety of cellular processes. Differences in peptide-binding specificity among Hsp70s have led to the hypothesis that peptide binding determines specific Hsp70 functions. Protein domains were identified that were required for two separate functions of a yeast Hsp70 family. The peptide-binding domain was not required for either of these specific Hsp70 functions, which suggests that peptide-binding specificity plays little or no role in determining Hsp70 functions in vivo.

Adenosine Triphosphatases↗
PubMed1997

Mitochondrial preprotein translocase.

Mitochondria import most of their proteins from the cytosol. Dynamic protein complexes in the mitochondrial outer and inner membranes are responsible for the specific recognition and membrane translocation of preproteins. The preprotein translocase of the outer mitochondrial membrane contains several import receptors and a general import pore. The preprotein translocase of the inner membrane consists of a channel interacting with preproteins in transit and an import motor that includes the matrix heat shock protein Hsp70. Acidic patches of import components are thought to guide the import of positively charged signal sequences (acid chain hypothesis). Energy input is derived from the inner membrane potential and ATP. Proteins in the mitochondrial matrix are required for proteolytic processing and folding of imported proteins. The dynamic nature of the membrane translocase permits sorting of preproteins at distinct stages of the import pathway.

Adenosine Triphosphatases↗
PubMed1996

Chaperones get Hip. Protein folding.

The discovery of a new co-chaperone, Hip, that interacts with Hsp70 underscores the complexity of the Hsp70 'chaperone machine' that mediates early steps of protein folding in cells.

Acute-Phase Proteins↗
PubMed1996

Acquired palsy of the oculomotor, trochlear and abducens nerves.

There have been few studies primarily concerned with the relative frequencies, aetiologies and prognoses of ocular motor palsies. Those published have emanated largely from neurological tertiary referral centres rather than primary ophthalmology departments. We have performed a retrospective study of all patients with acquired III, IV or VI cranial nerve palsy who were seen in the orthoptic department at Ninewells Hospital, Dundee, over the 9 year period from 1984 to 1992. A total of 165 cases were identified. VI nerve palsies accounted for the majority of patients (57%), with IV nerve palsies (21%) occurring more frequently than III nerve palsies (17%) and multiple palsies (5%). Thirty-five per cent of cases were of unknown aetiology and 32% of vascular aetiology. The incidence of sinister pathology-neoplasia (2%) and aneurysm (1%)-was surprisingly low. Fifty-seven per cent of all patients made a total recovery (in a median time of 3 months) and 80% made at least a partial recovery. The results are contrasted with those of previous studies and the value of associated symptoms and of further investigation in the assessment of these patients is discussed.

Abducens Nerve↗
PubMed1996

Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

Base Sequence↗
PubMed1996

SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.

Amino Acid Sequence↗
PubMed1996

Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo.

In order to analyze the in vivo role of the SSA class of cytosolic 70-kDa heat shock proteins (hsps) of Saccharomyces cerevisiae, we isolated a temperature-sensitive mutant of SSA1. The effect of a shift of mutant cells (ssa1ts ssa2 ssa3 ssa4) from the permissive temperature of 23 degrees C to the nonpermissive temperature of 37 degrees C on the processing of several precursor proteins translocated into the endoplasmic reticulum or mitochondria was assessed. Of three mitochondrial proteins tested, the processing of only one, the beta subunit of the F1F0 ATPase, was dramatically affected. Of six proteins destined for the endoplasmic reticulum, the translocation of only prepro-alpha-factor and proteinase A was inhibited. The processing of prepro-alpha-factor was inhibited within 2 min of the shift to 37 degrees C, suggesting a direct effect of the hsp70 defect on translocation. More than 50% of radiolabeled alpha-factor accumulated in the precursor form, with the remainder rapidly reaching the mature form. However, the translocation block was complete, as the precursor form could not be chased through the translocation pathway. Since DnaJ-related proteins are known to interact with hsp70s and strains containing conditional mutations in a dnaJ-related gene, YDJ1, are defective in translocation of prepro-alpha-factor, we looked for a genetic interaction between SSA genes and YDJ1 in vivo. We found that a deletion mutation of YDJ1 was synthetically lethal in a ssa1ts ssa2 ssa3 ssa4 background. In addition, a strain containing a single functional SSA gene, SSA1, and a deletion of YDJ1 accumulated the precursor form of alpha-factor. However, no genetic interaction was observed between a YDJ1 mutation and mutations in the SSB genes, which encode a second class of cytosolic hsp70 chaperones. These results are consistent with SSA proteins and Ydj1p acting together in the translocation process.

Adenosine Triphosphatases↗