FIGURE 4.

HRG-1 associates with the V-ATPase proton pump and enhances V-ATPase activity in yeast. A, HRG-1 interacts with V-ATPase c subunit. HEK-293T cells were cotransfected with HA empty vector and His-c subunit or HA-HRG-1 and His-c subunit. Transfected cells were maintained in complete media (CM), starved of serum (SS) for 20 h, or serum-starved (20 h) and stimulated with 100 ng/ml IGF-I. His-c subunit was immunoprecipitated (IP) using anti-His antibody, and coimmunoprecipitation of HA-HRG-1 was detected using anti-HA antibody. B, HRG-1 localizes with V-ATPase c subunit. HeLa cells transiently expressing HA-HRG-1 were cultured in complete media (CM) or serum-starved for 20 h (SS), costained with anti-HA antibody (red) or anti-c subunit antibody (green), and analyzed by epifluorescence microscopy. Merged images are shown, together with a zoom image of the insets. Arrows mark cells with overlapping fluorescence. C, HRG-1 interacts with V-ATPase holoenzyme. HEK-293T cells were transfected with HA (empty vector) or HA-HRG-1. Endogenous A subunit (A sub) was immunoprecipitated, and coprecipitation of HA-HRG-1 was examined by Western blot with anti-HA antibody. D, HRG-1 enhances V-ATPase activity in yeast. The yeast strain YPH500 was transformed with pGBK-T7-HRG-1 or pGBK-T7 vector alone. Top, vacuolar membranes were isolated, followed by Western blotting to detect subunit a (V₀ domain), subunit A (V₁ domain), and HRG-1. Bottom, vacuolar membranes were assayed for concanamycin A-sensitive ATPase activity (black bars) and concanamycin A-sensitive, ATP-dependent proton transport (white bars) as described in Ref. 27. Activities are expressed relative to vacuolar membranes isolated from the YPH500 strain expressing the pGBK-T7 vector alone. Error bars represent S.D. between vacuole preparations.