Properties of three isoforms of the 116‐kDa subunit of vacuolar H+‐ATPase from a single vertebrate species: Cloning, gene expression and protein characterization of functionally distinct isoforms in Gallus gallus
2000
Vacuolar H+‐ATPases (V‐ATPases) are involved in a wide variety of essential cellular processes. An unresolved question is how the cell regulates the activity of these proton pumps and their targeting to distinct cellular compartments. There is growing evidence for the presence of subunit diversity amongst V‐pumps, particularly regarding the 116‐kDa subunit (called the a subunit). We have cloned and characterized three isoforms (a1, a2 and a3) of this subunit from chicken. The amino‐acid sequences of these homologues are approximately 50% similar and their nucleotide differences indicate that they are products of distinct genes. The levels of mRNA expression of these isoforms was quantified by ribonuclease protection analysis. The a1 and a2 isoforms have a similar tissue distribution, with the highest level of mRNA expression in brain, an intermediate level in kidney and relatively low levels in liver and bone. In contrast, the highest level of expression of the a3 isoform is in bone and liver, with a moderate level in kidney, and the lowest level in brain. An antibody against the a1 isoform reacted with a 116 kDa protein in a brain V‐ATPase preparation that was not detected in bone or liver V‐ATPase preparations, whereas an antibody against the a3 isoform reacted with a 116‐kDa peptide in bone and liver, but not brain V‐ATPases preparations. The bone and brain V‐ATPases showed differential sensitivity to the inhibitors bafilomycin and (2Z,4E)‐5‐(5,6‐dichloro‐2‐indolyl)‐2‐methoxy‐N‐[4‐(2,2,6,6‐tetramethyl)piperidinyl]‐2,4‐pentadienamide. Thus, this work demonstrates the presence of structurally and functionally distinct V‐ATPases in a single vertebrate species.
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