title: Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.
abstract: ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.
authors.affiliation: From the Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Joel H
authors.initials: JH
authors.lastname: Otero
authors.affiliation: From the Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Beata
authors.initials: B
authors.lastname: Lizák
authors.affiliation: From the Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Feige
authors.affiliation: From the Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 linda.hendershot@stjude.org.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Hendershot
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism
chemical_list: C494648:DNAJB11 protein, human; D050956:HSP40 Heat-Shock Proteins; D011506:Proteins; D000251:Adenosine Triphosphatases
keywords: 70-kilodalton Heat Shock Protein (Hsp70); Chaperone DnaJ (DnaJ); Chaperone DnaK (DnaK); Endoplasmic Reticulum (ER); Endoplasmic Reticulum-associated Protein Degradation (ERAD); Protein Folding