118NISR RESEARCH GRANT
2003 Research Grant

Modification of Vacuolar Function and Physiology of Saccharomyces cerevisiae by Expressing Mammalian V-ATPases
Yoh WADADivision of Biological Science, Institute of Scientific and Industrial Research, Osaka University
Research aims
Vacuoles constitute a large compartment occupying aquarter to half of the cell volume. This large organelle has
function in maintaining cellular homeostasis by transporting small and macromolecules across the membrane. Vacuolar ATPases (V-ATPase) are a class of proton pumps
which hydrolyse ATP to ADPPi and transport protons
from one side of the membrane to another. The V-ATPase iscomposed of multiple subunits including A, B, C, D, E, F,G, H, a, c, c, c, and d. In Saccharomyces cerevisiae, each
subunit except a is encoded by a single gene, however, inhigher organisms like mouse, multiple loci encodes isoforms for each subunit. In addition, more variants can beproduced by an alternative splicing events. Therefore, themammalian exhibits a wide variety set of V-ATPase in theirsubunit compositions.
These different isoforms are expressed not only in specific tissues or cell types but their expressions are regulated
at developmental stages. Moreover, these subunit isoforms
determines the subcellular localization of V-ATPases.
These observations leads an argument that V-ATPases with
distinct isoforms may exhibits distinct catalytic or transport
profiles. In terms of applies sciences, mammalian isoforms
is a resource for altering the biochemical and physiological
function of ATPase.
In this research, we identified the multiple isoforms consituting the mammalian V-ATPase. We also established amethod for expressing the subunits of mammalian origin in
the yeast Saccharomyces cerevisiae: this system is quite
useful for determining the subunit characteristics.
Methods
Expression of mammalian V-ATPase subunits in yeast
cDNA candidates for each isoforms were identified by
EST database search using the corresponding yeast sequence as a probe. Full-length clones were obtained as EST
clones or reverse-transcription PCR products, and the coding regions were subcloned into a yeast expression plasmid
pKT10 which expresses exogeneous protein under a regulation of TDH3 promoter.
Characterization of yeast/mammalian hybrid V-ATPase on
the yeast vacuolar membrane
Yeast spheroplasts harbouring the expression plasmids
were generated and further incubated in YPD0.8 M sor-bitol. Vacuoles were prepared with discontinuous Ficoll
gradients and converted into vesicles. ATPase activity wasmeasured using a coupled spectrophotomeric assay. ATPdependent proton transport activity was measured byAMCA fluorescence quenching.
Preparation of Antibodies
Isoform-specific rabbit antibodies against synthetic peptides or bacterially expressed GST- or MBP-fusion proteins
were generated and purified with affinity columns conjugated with the corresponding recombinant proteins.
Results
Temperature dependent proton transport of mammalian Esubunit isoform
We have identified two novel mouse genes, Atp6e1 andAtp6e2, encoding testis-specific (E1) and ubiquitous (E2)
V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to
the start of meiosis, and is expressed specifically in roundspermatids in seminiferous tubules. Immunohistochemistrywith isoform-specific antibodies revealed that the V-ATPasewith E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature
sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartmentsof spermatocytes.
The E1 isoform exhibits 70% identity with the E2, andboth isoforms functionally complemented a null mutationof the yeast counterpart VMA4, indicating that they arebona fide V-ATPase subunits. The chimeric enzymes
showed slightly lower KmATP than yeast V-ATPase. Consistent with the temperature-sensitive growth of vma4-expressing E1 isoform, vacuolar membrane vesicles exhibited
temperature-sensitive coupling between ATP hydrolysis andproton transport.
Lung and kidney specific isoforms and their expression inyeast
We also identified two alternative splicing variants of C2subunit isoforms: C2-a, a lungspecific isoform containing a
46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C 2-a
is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma

119membranes of renal and intercalated cells. A chimeric yeast
V-ATPase with mouse the C2-a or C2-b isoform showed alower KmATP and lower proton transport activity than thatwith C1 or Vma5p (yeast C subunit).
Novel characteristic of V-ATPase: subunit rotation during
the enzyme reaction
The general structure of V-ATPase is similar to that of FATPase (ATP synthase) which also hydrolyses ATP andforms an electrochemical proton gradient. We showed continuous rotation of purified F-ATPase. Although the physiological role and structure of the two enzymes are different,
it has been proposed that V-ATPase has a rotary mechanismsimilar to that of F-ATPase. To examine this possibility, wefocused on the V-ATPase G subunit. Assuming that the Gsubunit rotates relative to the c ring of V-ATPase, we introduced a His tag and a biotin-binding domain to c and G, re-spectively, of the yeast enzyme. Upon ATP hydrolysis, weobserved continuous counter-clockwise rotation of an actin
filament connected to the G subunit of V-ATPase.
References1.

Hirata, T. et al., Subunit rotation of vacuolar-type proton pumping
ATPase: Relative rotation of the G as to c subunit, J. Biol. Chem.
278, 23714–23719, 2003
2. Sun-Wada, G. H. et al., Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung. J.
Biol. Chem. 278, 44843–44851, 2003
3. Sun-Wada, G. H. et al., Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits. Gene302, 147–153, 2003
4. Toyomura, T. et al., From lysosomes to plasma membrane: Localization of vacuolar type H-ATPase with the a3 isoform during osteoclast differentiation. J. Biol. Chem. 278, 22023–22030, 2003