Uncovering the prevalence, key biogenesis enzymes, and biological significance of archaeal lipoproteins.

Lipid-anchored proteins are integral components of cell surfaces. In bacteria, lipidation of proteins with a conserved lipobox motif ([L/V/I] [A/S/T/V/I] [G/A/S] [C]) is catalyzed by prolipoprotein diacylglyceryl transferase (Lgt). Although lipobox-containing proteins, or lipoproteins, are predicted to be abundant in several archaeal species, no archaeal homologs of Lgt have been identified, suggesting distinct lipidation enzymes evolved in archaea to accommodate their unique membrane lipids. Here, we predicted lipoprotein presence for all major archaeal lineages and revealed a high prevalence of lipoproteins across the domain Archaea. Using comparative genomics, we identified a comprehensive set of candidates for archaeal lipoprotein biogenesis components (Ali). Genetic and biochemical characterization in the archaeon Haloferax volcanii confirmed that two paralogous genes, aliA and aliB, are important for lipoprotein lipidation. Moreover, deletion of both genes led to a complete absence of diphytanylglyceryl thioether from lipoprotein extracts, revealing the chemical nature of lipid anchors in Hfx. volcanii lipoproteins. Disruption of AliA- and AliB-mediated lipoprotein lipidation caused severe growth defects, decreased motility, and cell-shape alterations, underscoring the importance of lipoproteins in archaeal cell physiology. Notably, AliA and AliB exhibit distinct, non-redundant enzymatic activities with potential substrate selectivity, uncovering a new layer of regulation in prokaryotic lipoprotein lipidation.