V1 domains can be found in the cytoplasm and not at the vacuole. In contrast, in vivo pulse-chase experiments have revealed early interactions between Vo and V1 subunits, to be specific, the a and B subunits, suggesting that subunits are added in a step-wise fashion to form a single complex in a concerted assembly process. V-ATPase evolution A relatively new technique called ancestral gene resurrection has shed new light on the evolutionary history of the V-ATPase. It has been shown how the V-ATPase structure of the ancestral form consisting of two different proteins evolves into the fungi version with three different proteins. The V-Type ATPase is similar to the archaeal (so called) A-Type ATP synthase, a fact that supports an archaeal origin of eukaryotes (like Eocyte Hypothesis, see also Lokiarchaeota). The exceptional occurrence of some lineages of archaea with F-type and of some lineages of bacteria with A-type ATPase respectively is regarded as a result of horizontal gene transfer. Regulation of V-ATPase activity V-ATPases are known to be specifically inhibited by macrolide antibiotics, such as concanamycin (CCA) and balifomycin A1. In vivo regulation of V-ATPase activity is accomplished by reversible dissociation of the V1 domain from the Vo domain. After initial assembly, both the insect Manduca sexta and yeast V-ATPases can reversibly disassemble into free Vo and V1 domains after a 2- to 5-minute deprivation of glucose. Reversible disassembly may be a general mechanism of regulating V-ATPase activity, since it exists in yeast and insects. Reassembly is proposed to be aided by a complex termed RAVE (regulator of -ATPase of vacuolar and endosomal membranes). Disassembly and reassembly of V-ATPases does not require new protein synthesis but does need an intact microtubular network. Human diseases Osteopetrosis Osteopetrosis is generic name that represents a group of heritable conditions in which there is a defect in osteoclastic bone resorption. Both dominant and recessive osteopetrosis occur in humans. Autosomal dominant osteopetrosis shows mild symptoms in adults experiencing frequent bone fractures due to brittle bones. A more severe form of osteopetrosis is termed autosomal recessive infantile malignant osteopetrosis. Three genes that are responsible for recessive osteopetrosis in humans have been identified. They are all directly involved in the proton generation and secretion pathways that are essential for bone resorption. One gene is carbonic anhydrase II (CAII), which, when mutated, causes osteopetrosis with renal tubular acidosis(type 3). Mutations to the chloride channel ClC7 gene also lead