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J Marszalek

Publications and source records attributed to J Marszalek.

At least 19 recordsLinked to original sources
PubMed2002

A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers.

Mitochondria contain a specialized system of molecular chaperones that plays a critical role in the biogenesis of Fe/S centers. This Hsp70:J-protein system shows many similarities to the system found in bacteria, but the precise role of neither chaperone system has been defined. However, evidence to date suggests an interaction with the scaffold protein on which a transient Fe/S center is assembled, and thus implies a role in either assembly of the center or its transfer to recipient proteins.

Escherichia coli↗
PubMed2001

Jac1, a mitochondrial J-type chaperone, is involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae.

A minor Hsp70 chaperone of the mitochondrial matrix of Saccharomyces cerevisiae, Ssq1, is involved in the formation or repair of Fe/S clusters and/or mitochondrial iron metabolism. Here, we report evidence that Jac1, a J-type chaperone of the mitochondrial matrix, is the partner of Ssq1 in this process. Reduced activity of Jac1 results in a decrease in activity of Fe/S containing mitochondrial proteins and an accumulation of iron in mitochondria. Fe/S enzyme activities remain low in both jac1 and ssq1 mutant mitochondria even if normal mitochondrial iron levels are maintained. Therefore, the low activities observed are not solely due to oxidative damage caused by excess iron. Rather, these molecular chaperones likely play a direct role in the normal assembly process of Fe/S clusters.

Aconitate Hydratase↗
PubMed2000

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1.

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

Aconitate Hydratase↗
PubMed1999

Dual role of the mitochondrial chaperone Mdj1p in inheritance of mitochondrial DNA in yeast.

Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA). Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions. The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p. At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p. However, under these conditions, intact [rho(+)] genomes were rapidly converted to nonfunctional [rho(-)] genomes which were stably propagated in an mdj1 deletion strain. We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature. In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.

DNA, Fungal↗
PubMed1998

Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli.

DnaA protein of Escherichia coli is a sequence-specific DNA binding protein required for the initiation of DNA replication from the chromosomal origin, oriC, and of several E. coli plasmids. At a moderate ionic strength, purified DnaA protein has a strong tendency to aggregate; the self-aggregate form is inactive in DNA replication. Binding of ATP or ADP to DnaA protein protected it from aggregation to maintain its replication activity. AMP or cyclic AMP had no protective effect. The molecular chaperone DnaK protected DnaA protein from aggregation with or without ATP. DnaJ and GrpE were not stimulatory. Chaperonins GroEL and GroES were also able to prevent aggregation but only in the presence of ATP. The studies presented here show that for DnaA protein to be active in the initiation of DNA replication, it must be prevented from forming a self-aggregate by the binding of adenine nucleotides, and/or by the action of molecular chaperones.

Adenine Nucleotides↗
PubMed1998

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Bacterial Proteins↗
PubMed1996

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.

Adenosine Triphosphate↗
PubMed1995

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1995

The requirement for molecular chaperones in lambda DNA replication is reduced by the mutation pi in lambda P gene, which weakens the interaction between lambda P protein and DnaB helicase.

During the initiation of lambda DNA replication, the host DnaB helicase is complexed with phage lambda P protein in order to be properly positioned near the ori lambda-lambda O initiation complex. However, the lambda P-DnaB interaction inhibits the activities of DnaB. Thus, the concerted action of bacterial heat shock proteins, DnaK, DnaJ, and GrpE, is required to activate the helicase. Wild-type phage lambda cannot grow on the E. coli dnaB, dnaK, dnaJ, and grpE mutants. However, lambda phage with a mutation pi in the lambda P gene, is able to produce progeny in these mutants as well as in the wild-type bacteria. Purified mutant lambda pi protein reveals a much lower affinity to DnaB than wild-type lambda P, and the lambda pi-DnaB complex is unstable. Also, a very low concentration of DnaK protein is sufficient to activate the helicase in a replication system based on lambda dv dsDNA. In that system, the mutant DnaK756 protein, inactive in the lambda P-dependent replication, revealed its activity in the lambda pi-dependent reaction. The lambda O-lambda P-dependent replication system based on M13 ssDNA efficiently replicates DNA in the absence of any chaperone protein, unless lambda P is substituted by the lambda pi mutant protein. Data presented in this paper explain why lambda pi phage is able to grow on wild-type and dnaK756 bacteria.

Bacterial Proteins↗
PubMed1995

Increasing neurofilament subunit NF-M expression reduces axonal NF-H, inhibits radial growth, and results in neurofilamentous accumulation in motor neurons.

The carboxy-terminal tail domains of neurofilament subunits neurofilament NF-M and NF-H have been postulated to be responsible for the modulation of axonal caliber. To test how subunit composition affects caliber, transgenic mice were generated to increase axonal NF-M. Total neurofilament subunit content in motor and sensory axons remained essentially unchanged, but increases in NF-M were offset by proportionate decreases in both NF-H and axonal cross-sectional area. Increase in NF-M did not affect the level of phosphorylation of NF-H. This indicates that (a) in vivo NF-H and NF-M compete either for coassembly with a limiting amount of NF-L or as substrates for axonal transport, and (b) NF-H abundance is a primary determinant of axonal caliber. Despite inhibition of radial growth, increase in NF-M and reduction in axonal NF-H did not affect nearest neighbor spacing between neurofilaments, indicating that cross-bridging between nearest neighbors does not play a crucial role in radial growth. Increase in NF-M did not result in an overt phenotype or neuronal loss, although filamentous swellings in perikarya and proximal axons of motor neurons were frequently found.

Amino Acid Sequence↗
PubMed1994

DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli.

DnaA protein of Escherichia coli acts in the initiation of chromosomal replication to bind to sequences in the chromosomal origin. On binding, it promotes the assembly of other replication proteins that serve to prime DNA replication and assemble the replication apparatus for bidirectional replication fork movement. A collection of monoclonal antibodies to DnaA protein have been produced, one of which is described here, that interferes with the action of DnaA protein in promoting formation of a prepriming complex. On the analysis of this process, the antibody appears to interfere with the physical interaction between DnaA and DnaB protein in the DnaB.DnaC complex. Cross-linking studies confirm that DnaA and DnaB proteins interact directly. These results provide the first direct evidence that one of the roles of DnaA protein is to act as a site for binding of DnaB protein to the DNA and perhaps orients DnaB helicase to account for the directionality of replication fork movement.

Antibodies, Monoclonal↗
PubMed1992

Defective replication activity of a dominant-lethal dnaB gene product from Escherichia coli.

dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shrimankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.

Bacterial Proteins↗
PubMed1991

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Adenosine Triphosphatases↗
PubMed1991

Regulatory properties of AMP deaminases from rat tissues.

1. Phosphocellulose column chromatography under double gradient conditions (phosphate and KCl) revealed two forms of AMP deaminase in rat heart and brain and a single form in the liver and skeletal muscle. 2. Kinetically all purified AMP deaminases were classified into two categories: those, which elute from the column at lower KCl and Pi concentrations, display low S0.5 value are only moderately affected by MgATP, MgGTP and Pi; and those which elute at higher KCl and Pi concentrations, display high S0.5 values and are strongly regulated by allosteric effectors. 3. Physiological significance of the occurrence of two kinetic forms of AMP deaminase in some tissues is discussed.

AMP Deaminase↗
PubMed1990

Regulation of platelet AMP deaminase activity in situ.

The regulation of platelet AMP deaminase activity by ATP, GTP and phosphate was studied in human platelets in situ, and in vitro after partial purification. In intact platelets, a similar 50% decrease in cytosolic ATP was induced by either glucose starvation or treatment with H2O2. During starvation, AMP deaminase was in the inhibited state, as ATP consumption was mostly balanced by the accumulation of AMP. During H2O2 treatment, however, the enzyme was in the stimulated state, as the AMP formed was almost completely deaminated to IMP. Cytosolic GTP fell by 40-50% in both starvation and H2O2 treatment. In contrast, intracellular phosphate was 4-5-fold higher in starved than in H2O2-treated cells. These data point to phosphate as the main regulator of AMP deaminase activity in situ. This conclusion was verified by kinetic analysis of partially purified AMP deaminase. At near-physiological concentrations of MgATP, MgGTP and phosphate, the S0.5 (substrate half-saturation constant) for AMP was 0.35 mM. Half-maximal stimulation by MgATP occurred at a concn. between 2 and 3 mM. This stimulation was antagonized by the inhibitory effects of phosphate (IC50 = 2.0 mM) and MgGTP (IC50 = 0.2-0.3 mM), which acted in synergism (IC50 is the concentration causing 50% inhibition). We conclude that the difference in adenylate catabolism between starved and H2O2-treated platelets is due to the distinct phosphate concentrations. During starvation, refeeding and H2O2 treatment, the values of the adenylate charge and the phosphorylation potential were kept closely co-ordinated, which may be effected by AMP deaminase.

AMP Deaminase↗