Characterisation of a Salmonella actin-binding protein

• Main Author: Hsu, C.-R.
• Published: University of Cambridge 2009
• Subjects: 579
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604683

<i>Salmonella</i> invasion protein C (SipC) and SipA bind actin directly. SipC inserts into the host cell plasma membrane from where it promotes effector delivery and is essential for <i>Salmonella</i> invasion. SipC-C (residues 200-409) directly nucleates actin polymerisation <i>in vitro</i> and induces cytoskeletal rearrangements at the leading edge when expressed in cells. By analysis a panel of SipC-C derivatives in transfected cells, a region spanning residues 377-409 was defined as necessary for actin reorganisation and leading-edge localisation. Residues 377-409 were sufficient to localise to zones of actin polymerisation in cells. SipC-N (residues 1-120) is sufficient to pair actin filaments <i>in vitro</i> and to induce elongated filopodia in cells. Two adjacent regions spanning residues 41-60 and 51-70 were identified as essential for inducing elongated filopodia. Corresponding proteins (SipC-N^Δ41-60) and SipC-N^Δ51-70) were expressed and purified. TEM of protein-F-actin mixtures revealed a deficiency in F-actin pairing. These data define the minimal N-terminal region required for filament pairing <i>in vitro</i> and corresponding filopodial elongation in cells. SipC derivatives containing equivalent deletions (SipC^Δ41-60 and SipC^Δ51-70) exhibited significantly reduced F-actin bundling activity. Derivatives with an extended deletion (SipC^Δ31-70) exhibited an equivalent defect. TEM of a C-terminal truncate (SipC^1-377) in complex with F-actin revealed an unanticipated defect in F-actin bundling, and a recombinant derivative containing a double deletion  (SipC^{1-377 Δ31-70}) was unable to bundle or pair F-actin. These data demonstrate that SipC-induced F-actin bundling requires unexpected interplay between the N-terminal and C-terminal domains. SipC is proposed to bundle F-actin via the N-terminal region to pair filaments and the C-terminal actin-binding domain to efficiently cluster filaments for formation of highly dense actin bundles.