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

J Biol Chem. 2002 May 3;277(18):15947-56. doi: 10.1074/jbc.M112214200. Epub 2002 Feb 8.

Abstract

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 co-localized 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 --> 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • COS Cells
  • Carrier Proteins / metabolism*
  • Chlorocebus aethiops
  • Cloning, Molecular
  • DNA Primers
  • Endoplasmic Reticulum / metabolism*
  • Escherichia coli
  • HeLa Cells
  • Heat-Shock Proteins / genetics*
  • Heat-Shock Proteins / metabolism
  • Humans
  • Melanoma, Experimental
  • Membrane Glycoproteins / genetics*
  • Membrane Glycoproteins / metabolism
  • Mice
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Biosynthesis
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Transfection

Substances

  • Carrier Proteins
  • DNA Primers
  • ERdj4 protein, mouse
  • Heat-Shock Proteins
  • Membrane Glycoproteins
  • Molecular Chaperones
  • Recombinant Proteins
  • Adenosine Triphosphatases
  • molecular chaperone GRP78