Late Endosomal Recycling of Open MHC‐I Conformers

With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty L d (eL d ) molecules, an open form of murine MHC‐I allele, in fibroblast‐like cells. Pulse labeling of cell surface eL d with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC‐I conformers. Kinetic modeling, using in‐house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eL d demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eL d distributed into pre‐degradative multivesicular bodies (MVBs), these LE subsets were not a source for eL d recycling. The LE recycling of eL d did not require Rab7 membrane domains, as demonstrated by Rab7‐silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eL d in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 232: 872–887, 2017. © 2016 Wiley Periodicals, Inc.