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Characterization of the Peripheral Stalk of the Vacuolar ATPase
Author(s) -
Bilbo Sarah,
Foreman Ryan,
Owegi Margaret,
Pappas Donald,
ParraBelky Karlett
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a472-e
Subject(s) - protein subunit , atpase , stalk , v atpase , membrane , mutant , atp synthase , chemistry , atp synthase gamma subunit , biochemistry , biophysics , enzyme , atp hydrolysis , biology , gene , horticulture
V‐ATPases are multisubunit ATP‐dependent proton pumps consisting of two domains: a peripheral V1 sector (subunits A‐H), which binds and hydrolyses ATP, and a membrane‐bound Vo sector (subunits a, c, c’, c”, d, and e), which forms the pore for proton transport. V1 and Vo subunits are hold together by one central stalk made of subunits D and F and one (or two) peripheral stalks made of C, E, G, H and the N‐end of subunit a. In order to better understand the function of the peripheral stalk subunit E, site‐directed mutations were performed and the analysis of these mutants is in progress. Our studies have shown that two residues at the N‐end of subunit E are essential for V‐ATPase function in vivo and in isolated vacuolar membranes. The mutation S78A, which destabilized subunit E and prevented assembly of V1 subunits at the membrane and I24N, which allowed stable expression of subunit E but cells lacked significant V‐ATPase activity. At the carboxyl end half of subunit E, the mutation T202A caused two‐fold increased Vmax and about two‐fold less of V1Vo assembly at the membrane; this mutation increased the specific activity of V1Vo by enhancing the kcat of the enzyme four‐fold. Reduced levels of V1Vo and Vo complexes at T202A membranes suggests that the balance between V1Vo and Vo was not perturbed, instead cells adjusted the amount of assembled V‐ATPase complexes in order to compensate for the enhanced activity.