Rabbit reticulocyte coated vesicles carrying the transferrin-transferrin receptor complex: I. Purification and partial characterization.
Coated vesicles bearing the transferrin-transferrin receptor complex were isolated from rabbit reticulocytes by freeze-thaw cell lysis, followed by differential centrifugation with pelleting of vesicles at 100,000 g. Electronmicroscopy demonstrated the vesicles to have the characteristic morphology of coated vesicles, including the appearance of triskelions. The protein composition of the vesicles as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis included transferrin, transferrin receptor, and proteins of apparent mol wt of approximately 180,000, 140,000, 100,000, and 47,000 daltons. The 180,000 and 100,000 mol wt proteins were identified as clathrin and coated vesicle assembly factor proteins, respectively, by Western blot analyses. The vesicles had a Mg2+-dependent ATPase with a specific activity of approximately 8.5 nmoles ATP converted/min/mg vesicle protein. The vesicles could acidify the intravesicular space, as evidenced by the stimulation of the Mg2+-ATPase by the protonophore FCCP. Reticulocytes appear to be an excellent source of coated vesicles and as such should provide a model for studying the endocytosis of transferrin and the steps of iron uptake that proceed in these vesicles.
D000136:Acid-Base Equilibrium; D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology; D000818:Animals; D008854:Microscopy, Electron; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D011990:Receptors, Transferrin / Q000378:metabolism*; D012156:Reticulocytes / Q000032:analysis / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000032:analysis / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism*
D011506:Proteins; D011990:Receptors, Transferrin; D014168:Transferrin; D000251:Adenosine Triphosphatases