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...

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