Localization and recycling of gp27 (hp24γ3):: Complex formation with other p24 family members
Localization and recycling of gp27 (hp24γ3):: Complex formation with other p24 family members
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DOI：
10.1091/mbc.10.6.1939
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发表时间：
1999-06-01
影响因子：
3.3
通讯作者：
Nilsson, T
中科院分区：
文献类型：
--
作者：
Füllekrug, J;Suganuma, T;Nilsson, T
我们在此报告了 gp27 (hp24 gamma(3)) 的表征，gp27 是分泌途径中小而丰富的跨膜蛋白 p24 家族的一种糖蛋白。免疫电子和共焦扫描显微镜显示，在稳定状态下，gp27 定位于高尔基体的顺侧。此外，在整个细胞质的 COPI 和 COPII 包被结构中检测到一些 gp27。这表明循环已通过三个方面得到证实。首先，15摄氏度的温度处理导致与顺行货物共定位的前高尔基体结构中类似于p27的积累。其次，布雷菲德菌素 A 治疗导致 gp27 重新定位到 KDEL 受体和 COPII 均呈阳性的外周结构中。第三，显性失活突变体 Sar1p 通过阻断 ER 输出将 gp27 捕获在内质网 (ER) 中。总之，这表明 gp27 在早期分泌途径中广泛循环。免疫沉淀和共表达研究进一步表明，很大一部分 gp27 存在于异源寡聚复合物中。 p24 家族的三个成员，GMP25 (hp24 alpha(2))、p24 (hp24 beta(1)) 和 p23 (hp24 delta(1))，以化学计量的量共沉淀。该异质复合物是特异性的。 p26 (hp24 gamma(4)) 的免疫沉淀无法共沉淀 GMP25、p24 或 p23。此外，发现很少的 p26 与 gp27 共沉淀。在 ER 输出水平上提出了复合物形成的功能要求。除非共表达其他 p24 家族蛋白，否则瞬时表达的 gp27 无法离开 ER。所附着的寡糖的比较表明gp27和GMP25的回收存在差异。只有极小部分的 GMP25 显示出复杂的寡糖。相比之下，所有 gp27 在稳态下均显示出中间酶和反式酶的修饰。我们从这些数据得出结论，gp27 的一部分作为与 GMP25、p24 和 p23 的异源寡聚复合物存在，并且这些复合物与单个 p24 蛋白处于动态平衡，以允许差异回收和分配。
We report here the characterization of gp27 (hp24 gamma(3)), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of similar to p27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively 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 alpha(2)), p24 (hp24 beta(1)), and p23 (hp24 delta(1)), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24 gamma(4)) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.