Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

Yochem, John; Uchida, Hidenobu; Sunshine, Melvin G.; Saito, Haruo; Georgopoulos, Costa; Feiss, Michael

doi:10.1007/bf00267593

Cited by 143 publications

(83 citation statements)

References 15 publications

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“…Chaperone Depletion-Two commonly used cellular assays for chaperone function of E. coli DnaK are bacteriophage propagation, which requires host Hsp70 to disassemble a phage protein complex in order to initiate phage DNA replication, and growth following protein misfolding and aggregation induced by heat shock (13,31,32). Previous studies report no defect in phage propagation upon truncation of the E. coli DnaK C terminus (33)(34)(35), and in agreement with these studies, we confirm that a plasmid expressing DnaK with the entire disordered C terminus removed (1-603) is fully capable of propagating in an E. coli dnaK-knock-out strain at 30°C (Fig.…”

Section: Dnak C-terminal Truncation Results In Cellular Defects Upon mentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide-and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.The ubiquitously distributed Hsp70 family of molecular chaperones shepherd newly synthesized polypeptide chains, protect cells from stress-induced protein aggregation, assist in protein translocation across membranes, and regulate assembly and disassembly of macromolecular complexes. These physiological functions are accomplished by a two-domain allosteric mechanism in which cycles of ATP binding and hydrolysis in the N-terminal nucleotide-binding domain (NBD) 2 control the binding and release of hydrophobic polypeptide segments in the substrate-binding domain (SBD) (1-3). Although we have gained an increasingly detailed picture of this allosteric transition in Hsp70 chaperones and modes of substrate interaction (4 -7), it is striking that there is much less known about the function of the extreme C-terminal unstructured region (Fig. 1). Binding of the C-terminal region of mammalian Hsc70 to substrate was suggested in earlier work (8, 9), and more recently, the C-terminal segment of eukaryotic cytoplasmic Hsp70s was found to contain a conserved tetratricopeptide repeat (TPR) domain interaction motif that mediates binding with Chip, Hip, or Hop co-chaperones that facilitate the assembly of a variety of Hsp70 complexes (3). Even though bacteria lack homologs of these Hsp70-interacting TPR domain proteins, many, including the extensively characterized E. coli Hsp70 DnaK, have a disord...

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Section: Dnak C-terminal Truncation Results In Cellular Defects Upon mentioning

confidence: 99%

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Smock

Blackburn

Gierasch

2011

Journal of Biological Chemistry

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“…This cycle is tightly controlled by cofactors that stimulate the rate of ATP hydrolysis, like members of the DnaJ family, or proteins that regulate nucleotide exchange, like GrpE in bacteria and mitochondria (12, 13), and Hip (14), Hop (15), and Bag-1 (16) in mammals. BiP undergoes the same ATP/ADP cycle to bind and release substrates as demonstrated by in vivo and in vitro studies (3, 17); however, very few mammalian BiP regulators have been identified.DnaJ was first identified as a cofactor of DnaK, the bacterial hsp70 homologue, which stimulated the ATPase activity of DnaK and helped replicate phage DNA in host cells (18). Since then a large number of DnaJ homologues have been identified and exist in all species and organelles.…”

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confidence: 99%

“…DnaJ was first identified as a cofactor of DnaK, the bacterial hsp70 homologue, which stimulated the ATPase activity of DnaK and helped replicate phage DNA in host cells (18). Since then a large number of DnaJ homologues have been identified and exist in all species and organelles.…”

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confidence: 99%

Identification and Characterization of a Novel Endoplasmic Reticulum (ER) DnaJ Homologue, Which Stimulates ATPase Activity of BiP in Vitro and Is Induced by ER Stress

Shen

Meunier

Hendershot

2002

Journal of Biological Chemistry

190

183

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The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 colocalized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His 3 Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation. The endoplasmic reticulum (ER)1 is the site of synthesis and maturation of secretory pathway proteins, which include resident proteins of the endocytic and exocytic organelles as well as surface and secreted proteins. Approximately one-third of all cellular proteins are translocated into the lumen of ER, which possesses a unique oxidizing and Ca 2ϩ -rich environment, where post-translational modification, folding, and oligomerization of nascent proteins occur. ER molecular chaperones and folding enzymes associate with the newly synthesized proteins to prevent their aggregation and help them fold and assemble correctly. Through a process called ER quality control, proteins that do not mature properly are retained in the ER and are eventually targeted for ER-associated degradation (ERAD) through the action of the chaperones (1).BiP, also known as GRP78, is the mammalian ER member of the Hsp70 family and was the first component of the ER quality control apparatus to be identified (2). Hsp70 family members exist in all organisms and in all organelles, where they aid in the folding and assembly of nascent proteins and prevent their aggregation during conditions of physiological stress (3, 4). Like other Hsp70 proteins, BiP plays an essential role in the biosynthesis of proteins. In addition, BiP maintains the permeability barrier of the ER translocon during early stages of protein translocation ...

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“…DnaK also functions in more specialized activities such as bacteriophage DNA replication (5,6). In all of these processes, DnaK is thought to interact transiently with unraveled segments of partially unfolded or denatured protein substrates.…”

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Characterization of a Lidless Form of the Molecular Chaperone DnaK

Buczynski

Slepenkov

Sehorn

et al. 2001

Journal of Biological Chemistry

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Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

Cited by 143 publications

References 15 publications

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

Identification and Characterization of a Novel Endoplasmic Reticulum (ER) DnaJ Homologue, Which Stimulates ATPase Activity of BiP in Vitro and Is Induced by ER Stress

Characterization of a Lidless Form of the Molecular Chaperone DnaK

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