ERdj3, a luminal ER DnaJ homologue, binds directly to unfolded proteins in the mammalian ER: identification of critical residues.
ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrated that ERdj3 binds directly to unfolded substrates. Secondary structure predictions suggested that the substrate binding domain of ERdj3 was likely to closely resemble Ydj1, a yeast cytosolic DnaJ family member, which was previously crystallized with a peptide bound to the C-terminal fragment composed of domains I, II, and III. Mutation of conserved residues in domain I, which formed the peptide binding site in Ydj1, affected ERdj3's substrate binding ability in mammalian cells and in vitro binding studies. Somewhat unexpectedly, we found that domain II, which is highly conserved among ERdj3 homologues, but very different from domain II of Ydj1, was also critical for substrate binding. In addition, we demonstrated that ERdj3 forms multimers in cells and found that the conserved carboxy-terminal residue phenylalanine 326 played a critical role in self-assembly. In vitro binding assays revealed that mutation of this residue to alanine diminished ERdj3's substrate binding ability, arguing that multimerization is important for substrate binding. Together, these studies demonstrate that the Ydj1 structure is conserved in another family member and reveal that among this group of DnaJ proteins domain II, which is not present in the closely related type II family members, also plays an essential role in substrate binding.
Jin
Yi
Y
Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105, USA.
Zhuang
Min
M
Hendershot
Linda M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D008156:Luciferases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D008156:Luciferases