Lee W, Ryu J, Hah J, Tsujita T, Jung C Y
Biophysics Laboratory, Veterans Affairs Medical Center, Department of Physiology and Biophysics, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14215, USA.
J Biol Chem. 2000 Apr 7;275(14):10041-6. doi: 10.1074/jbc.275.14.10041.
Facilitative glucose transporter isoform 4 (GLUT4) in rat adipocytes is largely sequestered in intracellular sites, and insulin recruits GLUT4 from these sites to the cell surface. The process is known to involve multiple intracellular compartments and associated proteins, many of which are yet to be identified. Recently, we purified three distinct insulin-sensitive intracellular GLUT4 compartments (G4T(L), G4H, and G4L) in rat adipocytes and unraveled several new resident proteins in these compartments. Here, we describe one of them, a 62-kDa protein, purified and identified as rat adipose tissue carboxyl esterase (p62/CE) by matrix-assisted laser desorption/ionization time of flight mass spectroscopy, reverse transcription-polymerase chain reaction, gene cloning, and immunological and enzymatic activity measurements. p62/CE in rat adipocytes was 80% cytosolic and 20% microsome-associated. It was found in all of the three insulin-sensitive intracellular GLUT4 compartments, and particularly enriched in G4T(L,) a compartment thought to represent GLUT4 endocytic vesicles. Significantly, an antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. Together, these findings suggest that p62/CE plays a key role in insulin-induced GLUT4 recruitment in rat adipocytes, probably by hydrolyzing acylglycerols or acyl-CoA esters to the respective free acids that are required for GLUT4 transport vesicle budding and/or fusion.
大鼠脂肪细胞中的易化性葡萄糖转运蛋白4（GLUT4）大部分被隔离在细胞内位点，胰岛素将GLUT4从这些位点募集到细胞表面。已知该过程涉及多个细胞内区室和相关蛋白，其中许多蛋白尚未被鉴定。最近，我们在大鼠脂肪细胞中纯化了三个不同的对胰岛素敏感的细胞内GLUT4区室（G4T(L)、G4H和G4L），并揭示了这些区室中的几种新的驻留蛋白。在此，我们描述其中一种，一种62 kDa的蛋白，通过基质辅助激光解吸/电离飞行时间质谱、逆转录-聚合酶链反应、基因克隆以及免疫和酶活性测量，纯化并鉴定为大鼠脂肪组织羧基酯酶（p62/CE）。大鼠脂肪细胞中的p62/CE有80%位于胞质溶胶中，20%与微粒体相关。在所有三个对胰岛素敏感的细胞内GLUT4区室中均发现了它，尤其在G4T(L)中富集，G4T(L)被认为代表GLUT4内吞小泡。值得注意的是，将抗p62/CE抗体引入大鼠脂肪细胞后，完全消除了宿主细胞中胰岛素诱导的GLUT4向质膜的募集，而不影响基础GLUT4的分布。总之，这些发现表明p62/CE在大鼠脂肪细胞胰岛素诱导的GLUT4募集中起关键作用，可能是通过将酰基甘油或酰基辅酶A酯水解为GLUT4运输小泡出芽和/或融合所需的相应游离酸来实现的。
