Distribution and structure of the vacuolar H + ATPase in endosomes and lysosomes from LLC-PK 1, cells

Abstract

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H + ATPase in LLC-PK 1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK 1 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 LLCPK 1 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.