Receptor-mediated endocytosis is sensitive to antibodies against the uncoating ATPase (hsc70).
We have investigated the functional role of the coated vesicle-uncoating ATPase (UA), a cognate heat shock protein (hsc70), in receptor-mediated endocytosis. A monoclonal antibody against bovine brain UA/hsc70 was generated that recognizes a 26 kDa proteolytic fragment harbouring the putative clathrin-binding site. In vitro, this antibody blocked the UA/hsc70-mediated release of clathrin from isolated coated vesicles (CVs). Upon microinjection into tissue culture cells, it specifically inhibited the heat shock-induced nuclear migration of UA/hsc70. This antibody also interfered with endocytosis of ligand-receptor complexes in injected cells. Two different systems were studied: the uptake of aggregated human IgG by BHK cells transfected with a human Fc receptor (FcRII), and the internalization of LDL by human fibroblasts. Injection of the monoclonal antibody in concentrations yielding approximately equal molar ratios of antibody to enzyme resulted in a reduction of endocytosis to 20-30% of control values, as seen by conventional light and confocal laser scanning microscopy, and by electron microscopy. In the transfected BHK cells, the endocytosed ligand remained associated with the labeling for clathrin and was not delivered to the endosomal compartment within the period expected from control serum- or non-injected cells. Thin sections revealed an accumulation of coated structures in the antibody-injected cells as compared to controls. Thus, our data show that UA is essential for normal receptor-mediated endocytosis, and is presumably involved in the uncoating of CVs preceding their fusion with endosomes.
Höning
S
S
Cell Biology Group, University of Bielefeld, FRG.
Kreimer
G
G
Robenek
H
H
Jockusch
B M
BM
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000502:physiology; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000502:physiology; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D011961:Receptors, Fc / Q000235:genetics / Q000502:physiology; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008077:Lipoproteins, LDL; D011956:Receptors, Cell Surface; D011961:Receptors, Fc