Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli

Abstract

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...

Trigger Factor Retards Protein Export in Escherichia coli

“…3, lane 6). This result is consistent with the observation that DnaK depletion exacerbates OmpA export defects produced by the disruption of secB (4,5). More significantly, overexpression of dnaK E171K did not block OmpA export in tigϪ/secBϪ cells (Fig.…”

“…Although the Bla targeting pathway(s) has never been clearly identified, previous studies have shown that a variety of physiological insults that increase the presence of abnormal proteins (and that likely induce the heat shock response) lead to Bla export defects (7,34). These and other results (5) suggest that Bla is targeted by one or more heat shock-regulated chaperones such as DnaK or GroEL. Inactivation of the SRP pathway has often been observed to delay Bla export, but as previously proposed (35) this effect is probably an indirect consequence of perturbing the function of heat shock proteins.…”

“…A subset of presecretory proteins are targeted to the E. coli IM by SecB, a nonessential "export-specific" chaperone found only in Gram-negative bacteria (3). Like other members of the hsp70 family, DnaK promotes protein export and can at least partially substitute for SecB (4,5). 2 GroEL may also play a role in protein sorting (6,7).…”

SecB is a bona fide generalized chaperone in Escherichia coli

“…Specifically, it has been reported that DnaK can substitute in the export of several SecB-dependent secretory proteins (35) and that the DnaK and GroEL chaperone machines can assist protein export in some other cases as well (36,37). Furthermore, similar to the DnaK system, SecB can prevent luciferase from aggregation and cooperate with DnaK͞DnaJ͞GrpE in the refolding of luciferase in vitro (13).…”

DnaK Promotes the Selective Export of Outer Membrane Protein Precursors in SecA-deficient Escherichia coli

“…SecB is a tetramer that interacts with its ligands via an ATP-independent kinetic partitioning mechanism (5,6). Several studies have indicated that highly abundant molecular chaperones such as DnaK and GroEL, which play essential roles in protein folding, can also maintain the transport competence of presecretory proteins (7)(8)(9)(10). DnaK and GroEL are structurally unrelated to SecB and bind polypeptides in an ATP-dependent cycle that is regulated by co-chaperones (11,12).…”