Popoff Vincent, Mardones Gonzalo A, Tenza Danièle, Rojas Raúl, Lamaze Christophe, Bonifacino Juan S, Raposo Graça, Johannes Ludger
Laboratoire Trafic et Signalisation, UMR144 Curie/CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
J Cell Sci. 2007 Jun 15;120(Pt 12):2022-31. doi: 10.1242/jcs.003020.
Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes.
先前的研究表明，网格蛋白、网格蛋白衔接蛋白AP1和epsinR以及retromer复合体在从早期/循环内体到反式高尔基体网络（TGN）的逆行分选过程中发挥作用。然而，这些蛋白质机制是在相同还是平行的途径上发挥作用仍不清楚。我们在此表明，网格蛋白和retromer亚基Vps26在超微结构水平上共定位于含有志贺毒素B亚基的早期/循环内体上，志贺毒素B亚基是一种经过充分研究的逆行运输货物。正如先前对网格蛋白的描述一样，我们发现干扰Vps26的表达会抑制志贺毒素B亚基向TGN的逆行运输。在这些条件下，将志贺毒素B亚基从含转铁蛋白的早期/循环内体中带出的内体小管似乎会稳定下来。这种情况与先前描述的低温孵育和网格蛋白缺失条件不同，在低温孵育和网格蛋白缺失条件下，发现志贺毒素B亚基的标记与转铁蛋白受体的标记重叠。此外，我们发现志贺毒素B亚基和转铁蛋白受体在网格蛋白缺失的细胞中靠近多囊泡内体积聚，这表明网格蛋白在液泡早期内体上启动逆行分选，然后需要retromer来处理逆行小管。因此，我们的研究结果确定了retromer复合体在志贺毒素B亚基逆行运输中的作用，并强烈表明网格蛋白和retromer在早期内体的连续逆行分选步骤中发挥作用。
