SecA is required for membrane targeting of the cell division protein DivIVA in vivo

The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localizes to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localization of DivIVA in these mutants. Surprisingly, DivIVA was delocalized, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localization depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localization is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

 Control experiments to confirm the cross-linking of SecA with DivIVA. (A) Pull-down experiment with His-DivIVA, DivIVA-GFP-His and GFP-His as bait proteins (strains BSN50, 3308 and BSN73, respectively). Pull-down fractions were analyzed by Western blotting for the presence of SecA (upper panel). To demonstrate that the bait proteins were pulled down efficiently, fractions were analyzed by Western blotting using DivIVA (middle panel) and GFP antisera (lower panel). (B) Pull-down experiment using SecA-His and GFP-His as bait proteins (strains BSN131, and 4097, respectively). Protein fractions were separated by SDS-PAGE (upper panel), and the presence of DivIVA was analyzed by Western blotting (lower panel). SecY, a known interaction partner of SecA, was pulled down with SecA-His. The protein band at around 55 kDa is elongation factor Tu (TufA), which appears to be an unspecific by-catch. The theoretical position of DivIVA on the SDS PAGE gel is indicated in brackets.

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