Vo domain generally transports protons against their own concentration gradient. Rotation of the Vo domain transports the protons in movement coordinated with the V1 domain, which is responsible for ATP hydrolysis. The Vo domain is hydrophobic and composed of several dissociable subunits. These subunits are present in the Vo domain to make this a functional proton translocase; they are described below. Subunit a/I The 116kDa subunit (or subunit a) and subunit I are found in the Vo or Ao complex of V- or A-ATPases, respectively. The 116kDa subunit is a transmembrane glycoprotein required for the assembly and proton transport activity of the ATPase complex. Several isoforms of the 116kDa subunit exist, providing a potential role in the differential targeting and regulation of the V-ATPase for specific organelles. The function of the 116-kDa subunit is not defined, but its predicted structure consists of 6–8 transmembranous sectors, suggesting that it may function similar to subunit a of FO. Subunit d/C Subunit d in V-ATPases, called subunit C in A-ATPases, is a part of the Vo complex. They fit onto the middle of the c ring, so are thought to function as a rotor. There are two versions of this subunit in eukaryotes, d/d1 and d2. In mammals, d1 (ATP6V0D1) is the ubiquitously expressed version and d2 (ATP6V0D2) is expressed in specific cell types only. Subunit c Similar to the F-type ATP synthase, the transmembrane region of the V-ATPase includes a ring of membrane-spanning subunits that are primarily responsible for proton translocation. Dissimilar from the F-type ATP synthase, however, the V-ATPase has multiple related subunits in the c-ring; in fungi such as yeast there are three related subunits (of varied stoichiometry) and in most other eukaryotes there are two. V-ATPase assembly Yeast V-ATPases fail to assemble when any of the genes that encode subunits are deleted except for subunits H and c". Without subunit H, the assembled V-ATPase is not active, and the loss of the c" subunit results in uncoupling of enzymatic activity. The precise mechanisms by which V-ATPases assembly are still controversial, with evidence suggesting two different possibilities. Mutational analysis and in vitro assays have shown that preassembled Vo and V1 domains can combine to form one complex in a process called independent assembly. Support for independent assembly includes the findings that the assembled Vo domain can be found at the vacuole in the absence of the V1 domain, whereas free