Lim S N, Bonzelius F, Low S H, Wille H, Weimbs T, Herman G A
Department of Pediatrics, Division of Gastroenterology and Nutrition, University of California, San Francisco 94143, USA.
Mol Biol Cell. 2001 Apr;12(4):981-95. doi: 10.1091/mbc.12.4.981.
Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5'-[gamma-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.
携带来自早期内体的循环质膜蛋白的囊泡尚未得到表征。利用转染了易化葡萄糖转运蛋白GLUT4的中国仓鼠卵巢细胞，我们鉴定出两类离散的、大小相似的小囊泡，它们源自早期内体。我们将这些内体后囊泡称为内吞小囊泡或ESV。一类ESV含有相当一部分转铁蛋白受体池，另一类含有GLUT4细胞总池的40%，并且富含胰岛素反应性氨肽酶（IRAP）。ESV含有细胞ubrevin和Rab4，但缺乏其他早期内体标记物，如EEA1或 syntaxin13。ESV依赖ATP、温度和胞质溶胶的形成已在体外从内体膜重建。鸟苷5'-[γ-硫代]三磷酸和新霉素，而非布雷菲德菌素A，在体外抑制ESV的出芽。一种识别GLUT4细胞质尾巴的单克隆抗体扰乱了GLUT4在体外靶向ESV，而不干扰IRAP或TfR的掺入。我们认为，胞质蛋白介导循环膜蛋白掺入离散的ESV群体，这些ESV作为载体囊泡，直接或间接地将货物从早期内体储存并运输到细胞表面。
