Wang X, Kumar R, Navarre J, Casanova J E, Goldenring J R
Institute for Molecular Medicine and Genetics, Departments of Medicine, Surgery and Cellular Biology and Anatomy, Medical College of Georgia and the Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912, USA.
J Biol Chem. 2000 Sep 15;275(37):29138-46. doi: 10.1074/jbc.M004410200.
Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.
极化上皮细胞通过受体介导的内吞、分选，然后再循环至合适的膜结构域的过程，维持基底外侧膜蛋白和顶端膜蛋白的极化分布。我们之前已经表明，小GTP结合蛋白Rab11a和Rab25与Madin-Darby犬肾细胞的顶端再循环系统相关。在此，我们利用野生型、显性负性和组成型活性突变体的诱导表达，直接比较Rab25和Rab11a在内吞后囊泡运输中的功能。我们发现，一种缺乏GTP结合能力的Rab11a突变体Rab11aS25N，能有效抑制转胞吞作用和顶端再循环，但未能抑制转铁蛋白再循环。同样，野生型Rab25或活性突变体Rab25S21V的表达均使IgA的顶端再循环和转胞吞作用受到超过50%的抑制，但对转铁蛋白的基底外侧再循环没有影响。有趣的是，GTP酶缺陷型突变体Rab11aS20V抑制了IgA从基底外侧到顶端的转胞吞作用，但对顶端或基底外侧再循环均无影响。这些结果表明，在极化的Madin-Darby犬肾细胞中，Rab11a和Rab25在转铁蛋白的基底外侧再循环中均不起作用，这与其他人最近的形态学观察结果一致。因此，转铁蛋白受体必须在将顶端定向货物分选到Rab11a/Rab25阳性的顶端再循环内体之前再循环至质膜。
