Abstract
Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.
Highlights
Vacuolar-type H؉-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target
After expressing the human gene in a mutant lacking the equivalent yeast gene, we evaluated four cell phenotypes: 1) growth ability on media containing a high-calcium concentration and elevated pH; 2) vacuolar acidification; 3) localization of the V1 and Vo subunits; and 4) endocytic recycling of Wsc1–3GFP (Fig. 1A)
Two strains expressing the human E2 (V1E2) or F (V1F) subunit showed partial growth rescue when expressed from the native promoter (Fig. 2A and Table 1), and they showed almost complete recovery when expressed from the TPI1 promoter (Fig. 2B and Table 1), suggesting that these human subunits partially complement the functions of the yeast subunits
Summary
Complementation of yeast genes encoding V-ATPase subunits with equivalent human genes. Among strains expressing the human gene from the native promoter, two strains expressing the human C1 (V1C1) or D (V1D) subunit completely recovered their ability to grow in medium containing 100 mM Ca2ϩ concentration or elevated pH (Fig. 2A and Table 1), suggesting functional conservation of these subunits between yeast and human. Two strains expressing the human E2 (V1E2) or F (V1F) subunit showed partial growth rescue when expressed from the native promoter (Fig. 2A and Table 1), and they showed almost complete recovery when expressed from the TPI1 promoter (Fig. 2B and Table 1), suggesting that these human subunits partially complement the functions of the yeast subunits. Expression of the VoA2 subunit partially rescued the growth of vph1⌬ cells only when expressed from the TPI1 promoter (Fig. 3, A and B, and Table 1), suggesting that the function of the a subunit is partially conserved between yeast and human
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