The Arf6 GTPase regulates membrane traffic between the plasma membrane (PM) and an endosomal compartment and influences the dynamics of the cortical actin cytoskeleton. In many cells this endocytic pathway is the route followed by PM proteins that lack clathrin localization sequences (such as MHCI, the major histocompatibility complex class I) and hence are endocytosed into cells independently of clathrin. After internalization, these membrane vesicles fuse with early endosomes containing clathrin-dependent cargo proteins. From here, proteins such as MHCI can either be trafficked on to late endosomes and lysosomes for degradation or recycled back to the PM along tubular endosomes that lack clathrin cargo. We have found that some lipid raft-associated proteins, those anchored to the membrane by a glycosylphosphatidyl inositol (GPI) moiety, also enter cells via this clathrin-independent mechanism and follow a pathway, either to degradation or recycling, similar to MHCI. We found that endocytosis of these GPI anchored proteins and MHCI is stimulated by active Arf6-GTP, is dependent upon PM cholesterol but is not dependent upon dynamin, a GTPase required for clathrin-dependent endocytosis. During our studies on recycling of MHCI back to the PM, we have uncovered an intricate coordination of Rab GTP-binding proteins in this process. Recycling of transferrin receptor, a typical clathrin-dependent cargo protein that is also recycled, requires the function of Rab11. The recycling of MHCI along distinct tubular structures requires the function of Rab11 and Rab22a. Rab22a is specifically required for the re-segregation of clathrin-independent cargo into these distinct tubular endosomes whereas Rab11 may function at a later step prior to PM fusion. The ability of cells to regulate separately the internalization and recycling of clathrin-dependent vs. clathrin-independent cargo proteins enables cells to differentially regulate these processes. Since this clathrin-independent endosomal system carries important membrane proteins that influence immune function and cell adhesion, such as MHCI and integrins, understanding how this pathway is regulated may be critical in disease states such as pathogen invasion and tumor cell metastasis.
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