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. 2001 Mar;2(3):244-8.
doi: 10.1093/embo-reports/kve042.

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria

B Py  1 , L Loiseau, F Barras
Affiliations

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria

B Py et al. EMBO Rep. 2001 Mar.

Abstract

The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE-GspF-GspL-GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE-GspF-GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE-GspF-GspL-GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.

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Figures

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Fig. 1. Schematic representation of the regions of GspF and GspM analysed in the yeast two-hybrid system. Schematic representation of the regions of GspF and GspM that have been fused at their N-terminus either to LexA or to the B42 activation domain. The number next to the boxes indicates the position of the residues that defined the domain studied. Hatched box, transmembrane region.
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Fig. 2. Co-immunoprecipitation of GspF, GspE and GspL with anti-GspF antibodies. Cells were solubilized in Triton X-100 and subjected to immunoprecipitation using anti-GspF antibodies. Immunoprecipitated material was analysed. Strains used were as follows: lane 1, DH5α; lane 2, DH5α(pK-EL); lane 3, DH5α(pCPP2215/pBADIK); lane 4, DH5α(pCPP2215/pK-EL). GspF, GspE and GspL were detected by immunoblotting with anti-GspF (A), anti-HA (B) or anti-VSV (C) antibodies, respectively. Visualization was by chemiluminescence (Amersham). GspF, GspE and GspL are indicated by an arrow.
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Fig. 3. Analysis of the GspE–GspF–GspL complex in the absence of GspF or GspL. A Triton X-100-soluble cell extract was subjected to immunoprecipitation using antibodies raised against either GspE (A–C) or GspF (D). GspE, GspL and GspF were detected in the immunoprecipitated material by immunoblotting using anti-HA (A), anti-VSV (B) or anti-GspF (C and D) antibodies, respectively. Strains used were as follows: lane 1, DH5α(pK-EL); lane 2, DH5α(pCPP2222/pK-E); lane 3, DH5α(pCPP2222/pK-EL); lane 4, DH5α(pK-E). Visualization was either by chemiluminescence (Amersham) (A and B) or colorimetry (C and D).
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Fig. 4. Model for the type II secretion machinery. The secretion machinery can be depicted as comprising three building blocks: the inner membrane platform (GspE–GspF–GspL–GspM, light stippling); the pseudopilus (GspG–GspH–GspI–GspJ–GspK, heavy stippling); and the gated outer membrane pore (GspC–GspD–GspS, black area). Cel5 is shown as an example of a secreted protein ‘en route’ to the cell exterior.
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References

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