TIGHT INSERTION OF CYTOCHROME-B(5) INTO LARGE UNILAMELLAR VESICLES

Cytochrome b5 spontaneously binds to liposomes in a 'loose', or transferable form, whereas in vivo b5 binds post-translationally to the ER in the 'tight' or nontransferable form. The mechanism of tight insertion is unknown, except that it does not require SRP or energy input. The present study shows that prolonged incubation of b5 with large unilamellar vesicles (LUVs) of phosphatidylcholine results in slow conversion of the loose to the tight form, with a halftime of days. However, the process is complex. When the b5-LUVs are depleted of loose b5, by transfer of b5 to Sonicated vesicles, the tight b5 is found to be concentrated to near saturating levels in a small fraction of the LUVs. If the LUVs devoid of tight b5 are recovered and then reincubated with fresh b5, the same slow transformation recurs. Apparently, a new population of vesicles, containing tight b5, is generated during the prolonged incubation with the protein. The b5-enriched LUVs contain about the same level of trapped sucrose as does the original vesicle preparation, indicating that vesicle integrity is maintained throughout the process. When fresh b5 is added to these tight b5-containing LUVs, all the freshly bound protein rapidly inserts (< 2 h) into the tight configuration. Apparently, the newly formed tight-b5/LUV vesicle population is 'insertion-active'. A model for these complex transformations is proposed.