The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c'' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c'' is not essential for assembly but is for functional coupling of the enzyme.