Hsp70 chaperones can potentially interact with one of several J domain-containing Hsp40 co-chaperones to regulate distinct cellular processes. However, features within Hsp70s that determine Hsp40 specificity are undefined. To investigate this question, we introduced mutations into the ER-lumenal Hsp70, BiP/ Kar2p, and found that an R217A substitution in the J domaininteracting surface of BiP compromised the physical and functional interaction with Sec63p, an Hsp40 required for ER translocation. In contrast, interaction with Jem1p, an Hsp40 required for ER-associated degradation, was unaffected. Moreover, yeast expressing R217A BiP exhibited defects in translocation but not in ER-associated degradation. Finally, the genetic interactions of the R217A BiP mutant were found to correlate with those of known translocation mutants. Together, our results indicate that residues within the Hsp70 J domain-interacting surface help confer Hsp40 specificity, in turn influencing distinct chaperone-mediated cellular activities.Most newly synthesized proteins require the assistance of molecular chaperones to fold into their native conformations. In addition, chaperones facilitate the assembly of macromolecular structures. To perform these functions, chaperones noncovalently bind to surface-exposed hydrophobic patches, thus preventing protein aggregation and, in some cases, providing a secure folding environment (1, 2). One family of chaperones, heat shock proteins of 70 kDa (Hsp70s), 3 forms a major component of the cellular folding and stress response machinery (3, 4). Hsp70s are quite abundant and can be found in nearly every cellular compartment in eukaryotes and in most prokaryotes. Not surprisingly, they catalyze a multitude of functions, including protein folding, transport, and degradation.Hsp70s contain a conserved N-terminal ATPase domain, followed by a less conserved substrate binding domain (SBD) and a variable C-terminal "lid." The lid is flexible and helps confine the substrate within the SBD. The Hsp70 ATP hydrolytic cycle correlates with substrate binding and release, such that the ADP-bound state of Hsp70s has a higher affinity for substrates than the ATP-bound state. Because the ATPase activity of Hsp70s is weak, Hsp40 co-chaperones are needed to accelerate ATP hydrolysis and promote maximal chaperone activity (5-7). Select Hsp40 family members can also deliver substrates to Hsp70s. The interaction between Hsp70s and Hsp40s is mediated by the Hsp40 J domain, an ϳ70-amino acid motif that forms a four-helix bundle. Notably, an invariant HPD motif between helices 2 and 3 of the J domain plays a critical role in facilitating Hsp70-Hsp40 interaction.In recent years, it has become clear that Hsp40s are more diverse than Hsp70s (8). For example, Escherichia coli encode three Hsp70s and six Hsp40s, whereas the budding yeast Saccharomyces cerevisiae expresses 14 Hsp70s and 22 Hsp40s. The diversity increases in higher eukaryotes, with humans expressing 20 Hsp70s and Ͼ50 Hsp40s. Moreover, a single Hsp70 can interact with m...