The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain

Abstract

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNAbinding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence-or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Dimerization properties of TraM, the antiactivator that modulates TraR‐mediated quorum‐dependent expression of the Ti plasmid tra genes

“…Many of the deletion mutants were not detectable or only poorly detectable by Western analysis of lysates from A. tumefaciens using anti‐TraM antiserum (Table 2). However, the chimeric cI′::TraM deletion proteins all were highly expressed in E. coli and were detectable at levels indistinguishable from the cI′ fusion to wild‐type TraM as assessed by Western analysis using an antibody directed against the λ cI headgroup (Toutain et al ., 2003) (data not shown).…”

“…Samples of purified proteins or of cross‐linked extracts were subjected to electrophoresis in 15% SDS polyacrylamide gels and separated TraM proteins were probed by Western blotting using murine polyclonal antisera raised against purified (His) 6 ‐TraM (Hwang et al ., 1999). Samples of the cI′ fusion proteins expressed in E. coli were separated by 12% SDS‐PAGE and probed using a polyclonal rabbit‐antiserum raised against the λ cI headpiece (the generous gift of Dr Jennifer Leeds, see Toutain et al ., 2003). Interactions between antibodies and TraM were visualized by using goat anti‐mouse or goat anti‐rabbit IgG conjugated to alkaline phosphatase (Sigma) with BCIP and NBT (BRL) as substrates (Sambrook et al ., 1989).…”

Repression of the RcsC‐YojN‐RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence

“…Precedents for the importance of these protein–protein interactions in regulation of the RcsC‐YojN‐RcsB system exist for the DjlA protein (Genevaux et al ., 2000). The interaction of DjlA with DnaK and the presence of an intact transmembrane dimerization domain are both required to activate the RcsC‐YojN‐RcsB phosphorelay (Toutain et al ., 2003). Work is in progress to determine whether IgaA undergoes homo– or heterotypic interactions with RcsC, YojN or other membrane proteins.…”

The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions

“…DjlA is a type III membrane‐anchored dimer that possesses a C‐terminal J‐domain, the only region of homology that it shares with either DnaJ or CbpA (Clarke et al ., 1996; 1997; Kelley and Georgopoulos, 1997a; Toutain et al ., 2003). DjlA is classed as a bona fide cochaperone for DnaK because a purified, truncated form of DjlA lacking the N‐terminal transmembrane domain can stimulate DnaK ATPase activity and assist DnaK in the reactivation of denatured firefly luciferase.…”