The secretin‐specific, chaperone‐like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

Abstract

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.

The C‐terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function

“…Manipulation of the PulS signal sequence generated soluble, periplasmic PulS which protected PulD from cleavage, but neither this protein nor a periplasmic MalE-PulS hybrid protein facilitated the proper localization of PulD to the outer membrane (Hardie et al, 1996b). These results suggest that PulD and PulS interact.…”

“…Cells were lysed in 4% SDS at 100ЊC, electrophoresed on a 10% SDS-PAGE gel, transferred to a PVDF membrane, and analysed by immunoblotting using pIV antisera. E. chrysanthemi and E. carotovora, which could account for the exchangeability of OutS and PulS (Hardie et al, 1996b). Analysis of all nine secretins using several algorithms that predict transmembrane ␤-strands for outer membrane proteins (Claros and von Heijne, 1994;Jahnig, 1990;Persson and Argos, 1994;Schirmer and Cowan, 1993) suggests that the C-terminal domain is hydrophilic and does not span the outer membrane (data not shown).…”

“…A periplasmic form of PulS (in which its lipoprotein signal sequence and the N-terminal cysteine of the mature protein have been replaced by the signal peptidase I signal sequence of pre-MalE) inhibits pullulanase secretion by causing mislocalization of PulD (Hardie et al, 1996b). Because an accessory protein is not known to be required for proper localization of wild-type pIV, we asked whether periplasmic PulS could protect pIV-PulD 65 from degradation and enable it to function in phage assembly.…”

“…10). Whether periplasmic PulS remained associated with the Triton-insoluble full-length pIV-PulD 65 that had aggregated could not be determined, because this material could only be solubilized by heating to 100ЊC in SDS, which disrupts the PulD-PulS complex (Hardie et al, 1996b 37 TBq mmol ¹1 ) was added to 0.1 ml of cells at 29 min after infection, and an equal volume of ice-cold 10% TCA was added at 30 min. The samples were centrifuged in a microcentrifuge for 10 min, and the pellets were rinsed with acetone, suspended in 25 l 3% SDS, and heated to 100ЊC.…”

“…Furthermore, although most of the Out proteins (homologues of the Pul proteins) from Erwinia chrysanthemi and Erwinia carotovora are interchangeable, the OutD proteins (PulD homologues) and the substrates to be exported are not (Lindeberg et al, 1996). Likewise, several of the Pul proteins can be replaced by Out proteins (Possot and Pugsley, 1997;Pugsley, 1996) without affecting pullulanase secretion, but OutD cannot substitute for PulD (Hardie et al, 1996b).…”