Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells.
Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.
Rodman
J S
JS
Department of Cell Biology and Physiology, Washington University School of Medicine, Jewish Hospital of St. Louis, Missouri 63110.
Stahl
P D
PD
Gluck
S
S
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D004848:Epithelium / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007553:Isotope Labeling; D007668:Kidney / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D011233:Precipitin Tests; D006180:Proton-Translocating ATPases / Q000032:analysis*; D013552:Swine; D014617:Vacuoles / Q000201:enzymology*
D006180:Proton-Translocating ATPases