Mutational and Bioinformatic Analysis of Haloarchaeal Lipobox-Containing Proteins

A conserved lipid-modified cysteine found in a protein motif commonly referred to as a lipobox mediates the membrane anchoring of a subset of proteins transported across the bacterial cytoplasmic membrane via the Sec pathway. Sequenced haloarchaeal genomes encode many putative lipoproteins and recent studies have confirmed the importance of the conserved lipobox cysteine for signal peptide processing of three lipobox-containing proteins in the model archaeon Haloferax volcanii. We have extended these in vivo analyses to additional Hfx. volcanii substrates, supporting our previous in silico predictions and confirming the diversity of predicted Hfx. volcanii lipoproteins. Moreover, using extensive comparative secretome analyses, we identified genes encodining putative lipoproteins across a wide range of archaeal species. While our in silico analyses, supported by in vivo data, indicate that most haloarchaeal lipoproteins are Tat substrates, these analyses also predict that many crenarchaeal species lack lipoproteins altogether and that other archaea, such as nonhalophilic euryarchaeal species, transport lipoproteins via the Sec pathway. To facilitate the identification of genes that encode potential haloarchaeal Tat-lipoproteins, we have developed TatLipo, a bioinformatic tool designed to detect lipoboxes in haloarchaeal Tat signal peptides. Our results provide a strong foundation for future studies aimed at identifying components of the archaeal lipoprotein biogenesis pathway.