Fig. 4. Localization of vacuolar H⁺-ATPase in phagosomes. Wild-type (A) and lvsA mutant cells (B) were allowed to phagocytose rhodamine-labeled yeast particles for 15 minutes. Cells were then fixed and the vacuolar H⁺-ATPase revealed by immunofluorescence. More vacuolar H⁺-ATPase was seen in phagosomes in lvsA mutant cells (B) than in wild-type cells (A). For electron microscopy, wild-type (C) and mutant lvsA cells (D) were allowed to phagocytose Klebsiella bacteria (K.a.) for 1 hour. Cells were then fixed and processed for immunoelectron microscopy using an antibody to vacuolar H⁺-ATPase and a secondary antibody coupled to 10 nm gold particles. Gold particles are indicated by arrows. The early phagosomes contain more vacuolar H⁺-ATPase in lvsA mutant cells than in wild-type cells (Table 1). A small number of late phagosomes (spacious and containing several bacteria) were also seen and were not considered in this study. Scale bar, 1 µm.