Localization and recycling of gp27 (hp24 gamma(3)): Complex formation withother p24 family members

We report here the characterization of gp27 (hp24 gamma(3)), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secr etory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In ad dition, some gp27 was detected in COPI- and COPII-coated structures through out the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of simi lar to p27 in pre-Golgi structures colocalizing with anterograde cargo. Sec ond, treatment with brefeldin A caused gp27 to relocate into peripheral str uctures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticu lum (ER) by blocking ER export. Together, this shows that gp27 cycles exten sively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24 alp ha(2)), p24 (hp24 beta(1)), and p23 (hp24 delta(1)), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Imm unoprecipitation of p26 (hp24 gamma(4)) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A funct ional requirement for complex formation was suggested at the level of ER ex port. Transiently expressed gp27 failed to leave the ER unless other p24 fa mily proteins were coexpressed. Comparison of attached oligosaccharides sho wed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showe d modifications by medial and trans enzymes at steady state. We conclude fr om these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibriu m with individual p24 proteins to allow for differential recycling and dist ributions.