Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12

Wada, Morimasa; Sekine, Kenji; Itikawa, Hiraku

doi:10.1128/jb.168.1.213-220.1986

Cited by 40 publications

(15 citation statements)

References 33 publications

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“…For example, Ydj1 has been shown to activate steroid hormone receptors and pp60 v-src (4,15). In addition, mutations in DnaJ or DnaK (Hsp70) in E. coli reduce the phosphorylation of threonyl-tRNA synthetase and glutamyl-tRNA synthetase (14,27) by mechanisms that have not yet been clarified. In mammalian cells, Hsp70 was found to associate with the heme-dependent serine/threonine kinase and to influence its activity during heat shock in response to the accumulation of abnormal proteins (17)(18)(19).…”

Section: Discussionmentioning

confidence: 99%

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Yaglom

Goldberg

Finley

et al. 1996

Molecular and Cellular Biology

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The G 1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34 CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34 CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34 CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34 CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.In Escherichia coli, mitochondria, and the cytosol of eukaryotic cells, members of DnaJ family of molecular chaperones function together with Hsp70s in protein folding and translocation across membranes (12). Recent studies have demonstrated that the chaperones are also essential for the rapid degradation of certain abnormal proteins in both prokaryotes and eukaryotes (16,24). These short-lived proteins appear to associate with DnaJ and other chaperones prior to their degradation (16,24). Since chaperones bind specifically to unfolded polypeptides, they may participate in the recognition of abnormal proteins by the degradative machinery. The present studies were undertaken to determine whether molecular chaperones may also be important in the degradation of regulatory proteins.To examine this possibility, we studied whether a DnaJ homolog in Saccharomyces cerevisiae, Ydj1, functions in the rapid ubiquitin-dependent breakdown of the G 1 cyclin Cln3. Cln3 is an important regulator of the Start checkpoint in the yeast cell cycle (7). Cln3 associates with and activates p34 CDC28 kinase in early G 1 phase, which leads to the synthesis of other G 1 cyclins (11, 24a, 25), thus allowing the transition from G 1 to S phase. This interaction occurs through the N-terminal segment of Cln3, which contains the cyclin box, a region conserved among different cyclins. Moreover, this N-terminal segment of Cln3 alone is sufficient for the stimulation of the kinase. p34CDC28 in turn phosphorylates Cln3, which triggers its ubiquitination, leading to the rapid degradation of Cln3 (8,28). This critical pho...

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Section: Discussionmentioning

confidence: 99%

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Yaglom

Goldberg

Finley

et al. 1996

Molecular and Cellular Biology

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“…Both DnaK and DnaJ act at the same step in in vitro A 0-, A P-dependent M13 or A dv DNA replication (18,20,21,42). The phosphorylation pattern of E. coli proteins is different in dnaK and dnaJ mutants but not in two other gro mutants, groEL and groES (40). The thermosensitive RNA synthesis of both dnaK and dnaJ mutants is relaxed in a relA background (34).…”

mentioning

confidence: 89%

Isolation and characterization of dnaJ null mutants of Escherichia coli

et al. 1990

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Bacteriophage A requires the X 0 and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block A growth at all temperatures and E. coli growth above 43°C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-TnlO transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30°C, although they blocked A DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42°C, and did not form colonies at 43°C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43°C. None of these suppressor mutations restored the ability of the host to propagate phage X. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaj-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage A growth at 42°C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein -is not absolutely essential for E. coli growth at temperatures up to 42°C under standard laboratory conditions but is essential for growth at 43°C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.Georgopoulos and Herskowitz (11) and Saito and Uchida (29) first described a class of mutations in Escherichia coli, called groP and grp mutations, respectively, which block A DNA synthesis and result in conditionally defective host DNA and RNA syntheses (29,30,39). Similarly, Sunshine et al. (33) isolated a bacterial mutation of the groP class, dnaJ259, which affects the growth of X and, to a lesser extent, P2 and also results in conditionally defective host DNA and RNA syntheses (39). It was later shown that dnaJ is closely linked to another groP gene, dnaK. The genes constitute an operon, the order being promoter-dnaK-dnaJ (30,43). Both genes have been sequenced (2,3,26), and the proteins encoded have been purified. DnaK is a 69.5-kilodalton protein that behaves like a monomer at low protein concentrations and possesses in vitro autophosphorylation and 5'-nucleotidase activities (4,46). DnaJ is a 44-kilodalton protein, behaves as a dimer under nondenaturing conditions, and is a nonspecific DNA-binding protein (47)...

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“…There is also evidence for an involvement of DnaK in cellular DNA synthesis (SAKAKIBARA 1988). Furthermore, participation of DnaK in reactions other than DNA replication has been reported, e.g., phosphorylation of tRNA synthetases (WADA et al 1986). The same subset of E. coli heat shock proteins seems to be involved in some of these processes, namely DnaK, DnaJ, and GrpE, which are all essential for bacterial growth.…”

Section: Dnak-the Prokaryotic Homologuementioning

confidence: 99%

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

Langer¹,

Neupert²

1991

Current Topics in Microbiology and Immunology

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Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12

Cited by 40 publications

References 33 publications

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Isolation and characterization of dnaJ null mutants of Escherichia coli

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

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Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12

Cited by 40 publications

References 33 publications

The Molecular Chaperone Ydj1 Is Required for the p34CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

Isolation and characterization of dnaJ null mutants of Escherichia coli

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

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The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation

The Molecular Chaperone Ydj1 Is Required for the p34^CDC28-Dependent Phosphorylation of the Cyclin Cln3 That Signals Its Degradation