Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruder granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.