Abstract
Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.
Highlights
Vacuolar Hϩ-ATPase (V-ATPase)1 proton pumps are present on vacuoles, lysosomes, endosomes, secretory vesicles, and Golgi of all eukaryotic cells where they maintain the acidic pH required for the multiple cellular processes achieved in these organelles [1,2,3]
Subunit E Mutagenesis—Amino acid sequence alignment of the V-ATPase subunit E from yeast, mammals, and insects shows a high degree of conservation both at the amino terminus (36% identity, residues 1–92) and carboxyl terminus (39% identity, residues 186 –233) (Fig. 1)
Alanine substitutions were performed at polar (Ser and Thr), charged (Asp and Lys), and aromatic (Tyr) residues at the amino terminal (Ser-2, Thr-6, Thr-9, and Ser-78) and carboxyl terminal (Tyr-160, Thr-202, Lys-230, and Asp-233) half of subunit E
Summary
Vacuolar Hϩ-ATPase (V-ATPase) proton pumps are present on vacuoles, lysosomes, endosomes, secretory vesicles, and Golgi of all eukaryotic cells where they maintain the acidic pH required for the multiple cellular processes achieved in these organelles [1,2,3]. Subunit D is functionally equivalent to ␥ of F1 and constitutes the rotating central stalk tightly associated with the proteolipid rotor in Vo. Comparable with subunits a and c from F0, the ring of proteolipid subunits (c, cЈ, and cЉ) and the Vo subunit a form the path for proton transport across the membrane. The structure and number of peripheral stalks could play a role regulating V-ATPase function by reversible disassembly, a mechanism exclusive of the V-type pump [35,36,37]. The regulator of ATPase of vacuolar and endosomal membranes (RAVE) complex and the glycolytic enzyme aldolase have been shown to interact with V-ATPase subunits in a glucose-dependent manner (17, 39 – 42)
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