We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways. Our previous finding that SecBdeficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway. Using depletion studies, we now demonstrate a requirement for GrpE in this pathway. In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and -lactamase), they are not the primary chaperones in this process.The DnaK, DnaJ, and GrpE proteins of Escherichia coli are heat shock proteins (Hsps) whose synthesis is induced upon exposure to high temperature. Both the structure and function of the Hsps are well conserved. The DnaK protein of E. coli has 50% identity with the Hsp70 family of proteins. Several eukaryotic homologs of DnaJ have been identified, and recently a homolog of GrpE was localized in yeast mitochondria (6,26).DnaK, DnaJ, and GrpE are involved in a large variety of cellular processes, including folding of nascent proteins (12, 43), DNA and RNA synthesis (7, 17), ribosome assembly (2), regulation of the heat shock response (30, 39), proteolysis (20, 37, 41), and , F, and P1 replication (16, 19, 44). The molecular role of each of these proteins has been investigated. DnaK binds to substrate molecules to influence the folding pathway, most generally by preventing off-pathway reaction but also possibly by stabilizing certain folding intermediates. DnaK has a weak ATPase activity, which is important in chaperone function. Both DnaJ and GrpE accelerate the ATPase cycle of DnaK, the former by promoting nucleotide hydrolysis and the latter by promoting nucleotide release. In addition, DnaJ binds independently to some substrates and may alter the substrate binding properties of DnaK (1,10,45).There is a great deal of evidence, particularly in eukaryotes, that the cell exploits the chaperone function of the Hsps to facilitate transport of molecules across membranes. Members of the Hsp70 family maintain proteins destined for secretion in a partially folded state thought to be a collapsed folding intermediate or a "molten globule" (15), which renders them competent for translocation into the mitochondrion, through the endoplasmic reticulum, or into other cellular compartments (14,35,42). Likewise, DnaK and DnaJ participate in protein secretion in E. coli cells. In this organism, SecB is a primary chaperone involved in secretion of many periplasmic and outer membrane proteins (23). In wild-type secB ϩ cells, mutations in dnaK, dnaJ, and grpE do not alter the secretion of SecB-dependent proteins. However, in secB-deficient strains, DnaK and DnaJ play a major role in secretion. In the absence of SecB, export of SecB-dependent proteins is not arrested but proceeds at a much slower rate. DnaK and DnaJ are required for this res...