High impact information on VPH1

• Here, we report that btn1-delta yeast strains have an abnormally acidic vacuolar pH in the early phases of growth [1].
• Vph1p is the 100 kDa membrane subunit of the yeast Saccharomyces cerevisiae V-ATPase, which together with other subunits, assembles into the V-ATPase in the ER, requiring the ER resident protein Vma22p [2].
• The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27 [3].
• Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride [4].
• On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p) [5].

Biological context of VPH1

• We show that the null V-ATPase mutants, as well as the vph1 triangle up one, markedly slow down endocytosis of the dye [6].
• Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases [7].
• DNA sequence analysis of the VPH1-SNF2 region on chromosome XV of Saccharomyces cerevisiae [8].
• Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes [9].
• As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification [10].

Anatomical context of VPH1

• High copy expression of STV1 partially restores vacuolar acidification in a delta vph1 mutant strain; solubilization and fractionation of membrane proteins from these vacuoles show that Stv1p co-purifies with bafilomycin A1-sensitive ATPase activity and with the 60- and 69-kDa V-ATPase subunits [11].
• We conclude that Stv1p is a functional homologue of Vph1p and suggest that Stv1p and Vph1p may be equivalent subunits for V-ATPases located on different organelles [11].
• Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes [9].
• Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V(0) subunit or an endoplasmic reticulum (ER) assembly factor [12].
• However, an additional effect of this phenotypic reversal is that activity of plasma membrane H(+)-ATPase is decreased further and vacuolar pH is increased further as btn1-Delta strains continue to grow [13].

Associations of VPH1 with chemical compounds

• Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 triangle up strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type [6].
• Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation [9].
• A recessive nuclear mutation, vph1-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine [10].
• Trypsin cleaved Vph1p at arginine 53 [14].
• A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity [15].

Physical interactions of VPH1

• Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p [9].
• Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase [16].
• Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction [14].
• An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles [17].

Other interactions of VPH1

• The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1 [9].
• In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function [18].
• We demonstrate that disruption of yeast VMA9 results in the failure of V1 and V0 V-ATPase subunits to assemble onto the vacuole and in decreased levels of the subunit a isoforms Vph1p and Stv1p [19].
• Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1) [14].
• We found that in vma21Delta cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly [17].

Analytical, diagnostic and therapeutic context of VPH1

• To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes [20].
• Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p [14].
• Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction [14].
• Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity [21].
• In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p [22].

References