COVI, JA; HAND, SC; Louisiana State University: Protein evidence for V-ATPase expression in Artemia franciscana gastrulae: Potential role in anoxia signaling.

Under anoxia, embryos of A. franciscana exhibit severe metabolic depression as they approach a state that is nearly ametabolic. For over 20 years it has been known that this state is accompanied by an extraordinarily large acidification of intracellular pH, which appears to play an essential role in the metabolic depression. However, the proximal mechanisms responsible for this critical pH shift remain unidentified. We hypothesize that dissipation of transmembrane proton gradients explains much of the observed intracellular acidification. Under such a scenario, transmembrane gradients would be established during aerobic development by a proton pump like the V-ATPase, dissipated under anoxia, and reestablished upon return to normoxia. We previously reported the expression profile of mRNA for the V-ATPase B-subunit. Here we report the first definitive evidence for the expression of V-ATPase protein subunits during preemergence development. Subcellular fractions were subjected to Western blot analysis using antibodies against the homologous enzyme from Manduca sexta. Results demonstrate the presence of B, E and G subunits. Comparison of the molecular mass estimated by electophoresis with that predicted from the deduced amino acid sequence derived from cDNA clones confirmed the identity of subunit B. The apparent lack of multiple isoforms for all identified subunits suggests the expression of a single V-ATPase complex. Further, the presence of these subunits within both heavy membrane and microsomal fractions implies cycling between the plasma membrane and vesicular pools. Histochemical localization of these subunits should offer greater insight into the functionality of the V-ATPase during the normoxia-anoxia transition. [Supported by DARPA grant N00173-01-1-G011]