The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli

In addition to promoting protein folding and translocation, molecular chape rones of Hsp70/DnaJ families are essential for the selective breakdown of m any unfolded proteins. It has been proposed that chaperones function in deg radation to maintain the substrates in a soluble form. In Escherichia coli, a nonsecreted alkaline phosphatase mutant that lacks its signal sequence ( PhoA Delta2-22) fails to fold in the cytosol and is rapidly degraded at 37 degreesC. We show that PhoA Delta2-22 is degraded by two ATP-dependent prot eases, La (Lon) and ClpAP, and breakdown by both is blocked in a dnaJ259-ts mutant at 37 degreesC. Both proteases could be immunoprecipitated with Pho A, but to a much lesser extent in the dnaJ mutant. Therefore, DnaJ appears to promote formation of protease-substrate complexes. DnaJ could be coimmun oprecipitated with PhoA, and the extent of this association directly correl ated with its rate of degradation. Although PhoA was not degraded when DnaJ was inactivated, 50% or more of the PhoA remained soluble. PhoA breakdown and solubility did not require ClpB. PhoA degradation was reduced in a thio redoxin-reductase mutant (trxB), which allowed PhoA Delta2-22 to fold into an active form in the cytosol. Introduction of the dnaJ mutation into trxB cells further stabilized PhoA, increased enzyme activity, and left PhoA com pletely soluble. Thus, DnaJ, although not necessary for folding (or prevent ing PhoA aggregation), is required for PhoA degradation and must play an ac tive role in this process beyond maintaining the substrate in a soluble for m.