The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The Cterminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine 3 glutamine substitution in the highly conserved HPD motif shared by all DnaJlike proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N-and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration-and temperaturedependent equilibrium. Heat shock proteins (HSPs) 1 are ubiquitous proteins found in all organisms and cell compartments. They are involved in cellular functions as varied as protein synthesis and proteolysis (1), stress tolerance (2), protein translocation into mitochondria (3) or the endoplasmic reticulum (4), and folding and assembly of proteins (5). Members of the HSP70 family are molecular chaperones that hold in common the ability to discriminate between unfolded polypeptide chains and native proteins. They participate in protein assembly by preventing aggregation due to hydrophobic interactions (6). HSP70 family members are highly conserved proteins, and share a structural organization in three domains: a 44-kDa N-terminal ATPase domain (7-9), an 18-kDa C-terminal peptide-binding domain (10, 11), and a less conserved C-terminal tail whose function and complete three-dimensional structure are unknown. The ATPase activity of HSP70 proteins is enhanced upon binding to synthetic peptides (12, 13). The size and nature of peptidic substrates required to fully stimulate the ATPase activity of HSP70 proteins have been determined to be 7-residue-long peptides, rich in aliphatic amino acids (14 -16). Detailed enzymological studies on monomeric, recombinant bovine Hsc70 revealed that binding of peptides increases the rate of ADP and inorganic phosphate release leading to an increase in the rate of ATP hydrolysis (17). In the same study, Takeda and McKay observed that the Mg.ADP.Hsc70 form has higher affinity for peptide than the Mg.ATP.Hsc70 form, in agreement with studies on Escherichia coli DnaK (18,19) and bovine brain Hsc70 (20). It was recently demonstrated that ATP-bound HSP70 proteins have high on/off rates of binding/release of substrates, whereas ADP-bound chaperones exhibit higher affinity for peptidic substrates through slower off rates (reviewed in Ref. 21). Many HSP70 proteins are also regulated by accessory proteins. A well studied system is the E. coli DnaK chaperone, which is regulated by the nucleotide exchange factor GrpE, which increases the rate of ADP/ATP exchange (22, 23) and by the highly conserved DnaJ, which increases the rate of ␥-phosphate cleavage (16,22). In addition to the different conformations HSP70 proteins adopt in the presence of substrates, they also self-associate into multiple oligomeric species that interconvert with each other (24 -26). In the bacterial system, addition of the dimeric cofactor GrpE to heterogeneous multimeric DnaK results in a slow conversion of oligomers into monomers and stabilization of (GrpE) 2 ⅐DnaK complexes (25). Mammalian Hs...

The murine tumor cell DnaJ-like protein 1 or MTJ1/ ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620 -19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with ␣ 1 -antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nM. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions. The rough endoplasmic reticulum (ER)1 is the primary site for the synthesis and maturation of secreted and membrane proteins. At this site molecular chaperones and their associated enzymes promote the folding and assembly of newly synthesized polypeptides (1, 2). Only native proteins leave the ER to enter the vesicular secretory pathway and reach their appropriate destinations. Misfolded or aberrant proteins are transported back to the cytosol, then transferred to the 26 S proteasome in a process termed ER-associated protein degradation (3-6).The molecular chaperone immunoglobulin heavy chain-binding protein (BiP)/GRP78, a member of the Hsp70 family resident of the ER, is involved in many cellular processes that include the translocation of newly synthesized polypeptides across the ER membrane, participation in their folding and maturation, assisting in refolding and renaturation, targeting misfolded proteins to the cytosol for proteasomal degradation, maintaining selectivity of the ER membrane by closing the translocon pore, as well as regulating calcium homeostasis (7-10). ATPase activity of BiP is required for most of these processes (11,12). In the ATP-bound form BiP binds to unfolded substrates with low affinity. The hydrolysis of ATP to ADP induces conformational changes that stabilize the BiPunfolded substrates co...

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