| The vacuolar ATPase (V-ATPase) is a macromolecular complex responsible for the acidification of intracellular organelles in Eukaryotes. In the yeast Saccharomyces cerevisiae, the V-ATPase is composed of 14 unique subunits and is divided into two distinct subcomplexes, V1 and V0. The peripheral V1 subcomplex contains the machinery for the hydrolysis of ATP and couples this activity to proton translocation, whereas the V 0 sector is responsible for proton translocation.; The V0 subcomplex is composed of five unique integral membrane proteins, subunit a, e, c, c', c'', and one peripheral membrane protein, subunit d. The small c subunits c and c' were shown to contain four transmembrane helices with the N and C termini located within the lumen of the vacuole. Subunit c'' was shown to contain five transmembrane helices with the N-terminus located on the cytoplasmic face of the vacuole and the C-terminus found within the lumen.; The V1 subcomplex is composed of eight subunits, A, B, C, D, E, F, G, and H. In S. cerevisiae, cells lacking the H subunit are still able to produce an assembled but non-functional V-ATPase complex on the membrane of the vacuole, hinting that the H subunit may play a key regulatory role in V-ATPase function. Through the use of truncation mutants of the subunit H gene, it was determined that the C-terminal portion of H is required for the activation of the V-ATPase complex. Specifically, amino acids 180-223 is required for activation but not binding, whereas the C-terminal domain (amino acid 353-478) appears to be required for binding to the V-ATPase. Using hetero-bifunctional chemical crosslinkers, we found that subunit H directly interacts with the B subunit and a 50 kD protein that remains to be identified.; This dissertation includes both my previously published and my co-authored materials. |
