Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements.
Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.
Naslavsky
Naava
N
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Weigert
Roberto
R
Donaldson
Julie G
JG
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018957:CD59 Antigens / Q000032:analysis / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D002966:Clathrin / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D005372:Filipin / Q000494:pharmacology; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D014168:Transferrin / Q000032:analysis / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000502:physiology; D020696:rab5 GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D018957:CD59 Antigens; D002966:Clathrin; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D014168:Transferrin; D005372:Filipin; D002784:Cholesterol; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020696:rab5 GTP-Binding Proteins