Nagamatsu S, Nakamichi Y, Watanabe T, Matsushima S, Yamaguchi S, Ni J, Itagaki E, Ishida H
Departments of Biochemistry, Clinical Pathology, Internal Medicine (III), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
J Cell Sci. 2001 Jan;114(Pt 1):219-227. doi: 10.1242/jcs.114.1.219.
Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.
细胞ubrevin是在多种组织中表达的整合膜蛋白，通常定位于再循环小泡中。在此，我们研究了一种与细胞ubrevin相关的肽——内体ubrevin在胰腺β细胞中的细胞定位及其在胰岛素和γ-氨基丁酸（GABA）外排-内吞作用中的意义。免疫细胞化学显示内体ubrevin与管状小泡结构相关，这些结构在胰岛素瘤MIN6细胞中与早期内体抗原（EEA）-1标记的早期内体共定位。为了确定内体ubrevin的细胞定位，我们将绿色荧光蛋白（GFP）附加到内体ubrevin上，并将融合蛋白导入MIN6细胞。通过共聚焦激光显微镜观察GFP-内体ubrevin的亚细胞定位。基于表达的GFP-内体ubrevin和内吞的德克萨斯红（Tx-R）标记的转铁蛋白受体的共定位研究以及用抗EEA1抗体进行的免疫细胞化学显示，内体ubrevin优先定位于早期内体。然后，我们研究了内体ubrevin在胰腺β细胞胰岛素和GABA外排中的功能作用。内体ubrevin过表达增加了MIN6细胞释放的GABA量；相反，它将大鼠胰岛、MIN6和INS1-D细胞中葡萄糖刺激的胰岛素释放降低到对照水平的约50%。体外和体内结合研究均表明内体ubrevin与 syntaxin 1结合。最后，使用荧光探针FM4-64，发现内体ubrevin过表达加速了小泡再循环。我们得出结论：（1）内体ubrevin定位于胰腺β细胞的早期内体中；（2）内体ubrevin在胰腺β细胞胰岛素和GABA的外排-内吞作用中发挥生理作用，可能是通过内吞小泡与内体之间的相互作用。
