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Coimmunoprecipitation is a powerful and commonly used method to identify protein-protein interactions in a physiological context. Here, we report a coimmunoprecipitation protocol that was adapted and optimized for the haloarchaeon Haloferax volcanii to identify interacting partners to the LonB protease. This protocol includes the in vivo cross-linking of H. volcanii proteins using two different crosslinker agents, dithiobis(succinimidyl propionate) and formaldehyde, followed by immunoprecipitation with anti-LonB antibody conjugated to Protein A - Sepharose beads. Tryptic on-bead protein digestion was performed combined with Mass Spectrometry analysis of peptides for the identification and quantification of LonB ligands.

Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a ‘recognition determinant’ for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.