Evidence for covalent attachment of diphytanylglyceryl phosphate to the cell-surface glycoprotein of Halobacterium halobium

In a previous study, we demonstrated the occurrence of novel proteins modified with a diphytanylglyceryl group in thioether linkage in Halobacterium halobium (Sagami, H,, Kikuchi, A., and Ogura, K, (1995) J. Biol, Chem. 270, 14851-14854), In this study, we further investigated protein isoprenoid modification in this halobacterium using several radioactive tracers such as [H-3]geranylgeranyl diphosphate. One of the radioactive bands observed on SDS-polyacrylamide gel electrophoresis corresponded to a periodic acid-Schiff stain-positive protein (200 kDa), Radioactive and periodic acid-Schiff stain-positive peptides (28 kDa) were obtained by trypsin digestion of the labeled proteins. The radioactive materials released by acid treatment of the? peptides showed a similar mobility to dolichyl (C-55) phosphate on a normal-phase thin-layer plate. However, radioactive hydrolysates obtained by acid phosphatase treatment co-migrated not with dolichol (C55-65), but with diphytanylglycerol on both reverse- and normal-phase thin-layer plates, The mass spectrum of the hydrolysate was also coincident with that of diphytanylglycerol, The partial amino acid sequences of the 28-kDa peptides were found in a fragment (amino acids 731-816) obtainable by trypsin cleavage of the known cell-surface glycoprotein of this halobacterium, These results indicate that the cell-surface glycoprotein (200 kDa) is modified with diphytanylglyceryl phosphate.