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Abstract

In the absence of a high-resolution structure for the vacuolar H+-ATPase, a number of approaches can yield valuable information about structure/function relationships in the enzyme. Electron microscopy can provide not only a representation of the overall architecture of the complex, but also a low-resolution map onto which structures solved for individually expressed subunits can be fitted. Here we review the possibilities for electron microscopy of the Saccharomyces V-ATPase and examine the suitability of V-ATPase subunits for expression in high yield prokaryotic systems, a key step towards high-resolution structural studies. We also review the role of experimentally-derived structural models in understanding structure/function relationships in the V-ATPase, with particular reference to the complex of proton-translocating 16 kDa proteolipids in the membrane domain of the V-ATPase. This model in turn makes testable predictions about the sites of binding of bafilomycins and the functional interactions between the proteolipid and the single-copy membrane subunit Vph1p, with implications for the constitution of the proton translocation pathway.

Building the Stator of the Yeast Vacuolar-ATPase

“…In addition, it is observed that this association is mutually beneficial to the stabilization of both subunits as shown from the reduced proteolysis of both subunits E and G. This is consistent with the in vivo stabilization of subunit E upon complex formation with subunit G (16). Considering that subunit G alone is misfolded, this increased stabilization can be associated with a more structured polypeptide chain, implying that the interaction with subunit E stabilizes a folded conformation of subunit G. This unfolded state of the isolated subunit G has been reported previously (37,45,46), and similar behavior in terms of aggregation and stability was observed for some mammalian orthologs of subunit G that share high sequence identity, suggesting that this assisted folding might be a general feature of the subunit G.…”

“…Expression at 19°C in BL21 (DE3) E. coli cells made it possible to obtain good yield of soluble fusion protein (25-50 mg/liter) in contrast to studies in which subunit E could be expressed only in inclusion bodies (37) or with the use of a special osmoregulated system (31). As seen in Fig.…”

Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F1FO ATP synthase

“…It has been shown that transmembrane domain 1 of V 0 V 1 -ATPase c-subunits faces the center of the V 0 rotor structure and lines a water-accessible pore structure [134,135] V 0 c-subunits have been shown to be involved in formation of mega-channels in gap junctions between cells [136]. Therefore pore-forming ability is an important feature of all homologous c-subunits which share similar amino acid sequence [118].…”