Role of Vacuolar Acidification in Protein Sorting and Zymogen Activation: a Genetic Analysis of the Yeast Vacuolar Proton-Translocating ATPase

Abstract

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

Localization of Subunits D, E, and G in the Yeast V-ATPase Complex Using Cysteine-Mediated Cross-Linking to Subunit B

“…In yeast subunit B is encoded by the VMA2 gene. The yeast VMA2 -deleted strain, SF838-5AV1 ( vat2 -Δ 1::LEU2 ), and pCY41 ( VMA2 in pBluescript) have been described previously ( , ) and were kind gifts from Dr. Patricia Kane, Upstate Medical University.…”

“…Transformation of yeast and selection of transformants on Uraplates were carried out as described previously (41). The mutants were then tested for growth on pH 7.5 or 5.5 yeast extract-peptone-dextrose plates buffered with 50 mM KH 2 PO 4 and 50 mM succinic acid (44).…”

Mutational Analysis of the Catalytic Subunit of the Yeast Vacuolar Proton-Translocating ATPase

“…For the integrations and subsequent biochemical characterization, wild-type strain SF838-5AR (MATR, leu2-3,112, ura3-52, ade6;Ito et al, 1983) and SF838-5AR vma1∆-8 (same as SF838-5AR with Vma1∆::LEU2; Kane et al, 1990) were used. Yeast cells were grown in rich medium (yeast extractpeptone-dextrose; YEPD) or supplemented synthetic dextrose medium (SD) (Sherman et al, 1982); preparation of buffered medium for selection of ATPase mutants was done as described in Yamashiro et al (1990).…”

“…The regions of the gene encoding the 69 kDa subunit are 62-73% identical to the catalytic subunits of other V-type ATPases and also show significant homology with the catalytic subunits of the F-type ATPases, particularly in regions previously implicated in ATP binding and catalysis (Zimniak et al, 1988;Bowman et al, 1988). Deletion of the chromosomal copy of the VMA1 gene results in complete loss of vacuolar acidification and ATPase activity in isolated vacuoles (Hirata et al, 1990;Kane et al, 1990) and also yields a set of growth phenotypes characteristic of mutants lacking vacuolar ATPase activity, including the ability to grow in medium buffered to pH 5, but not medium buffered to pH 7.5, and sensitivity to high (g100 mM) calcium concentrations (Nelson & Nelson, 1990;Ohya et al, 1991;Yamashiro et al, 1990). Precise deletion of the VDE- encoding portion of the VMA1 gene does not result in any of these phenotypes, indicating that the 69 kDa subunit can be synthesized in an active form in the absence of protein splicing (Kane et al, 1990).…”

The Toxoplasma plant‐like vacuolar compartment (PLVAC)

“…These lysosome-like organelles are acidified by the V-H + -ATPase complex (Hayashi et al, 2000;Saliba et al, 2003;Stasic et al, 2019;Yamashiro et al, 1990) and in addition in S. cerevisiae an NHE family of Na + / H + exchanger (Nhx1), which also transports K + , was shown to regulate both luminal and cytoplasmic pH and control vesicle trafficking out of the endosome (Brett et al, 2005). In addition, the PLVAC and DV express a V-H + -PPase, for proton pumping into the vacuole (Liu et al, 2014;Luo et al, 1999;Saliba et al, 2003).…”