Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.