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Vma8p-GFP Fusions Can Be Functionally Incorporated into V-ATPase, Suggesting Structural Flexibility at the Top of V1
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton campus, Victoria 3800, Australia
* Author to whom correspondence should be addressed.
Received: 7 June 2011; in revised form: 4 July 2011 / Accepted: 13 July 2011 / Published: 20 July 2011
Abstract: The vacuolar ATPase (V-ATPase) complex of yeast (Saccharomyces cerevisiae) is comprised of two sectors, V1 (catalytic) and VO (proton transfer). The hexameric (A3B3) cylinder of V1 has a central cavity that must accommodate at least part of the rotary stalk of V-ATPase, a key component of which is subunit D (Vma8p). Recent electron microscopy (EM) data for the prokaryote V-ATPase complex (Thermus thermophilus) suggest that subunit D penetrates deeply into the central cavity. The functional counterpart of subunit D in mitochondrial F1FO-ATP synthase, subunit γ, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit g, we probed the location of the C-terminus of Vma8p by attachment of a large protein adduct, green fluorescent protein (GFP). We found that truncated Vma8p proteins lacking up to 40 C-terminal residues fused to GFP can be incorporated into functional V-ATPase complexes, and are able to support cell growth under alkaline conditions. We conclude that large protein adducts can be accommodated at the top of the central cavity of V1 without compromising V-ATPase function, arguing for structural flexibility of the V1 sector.
Keywords: V-ATPase; Vma8p; yeast
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Nowakowski, S.; Mijaljica, D.; Prescott, M.; Devenish, R.J. Vma8p-GFP Fusions Can Be Functionally Incorporated into V-ATPase, Suggesting Structural Flexibility at the Top of V1. Int. J. Mol. Sci. 2011, 12, 4693-4704.
Nowakowski S, Mijaljica D, Prescott M, Devenish RJ. Vma8p-GFP Fusions Can Be Functionally Incorporated into V-ATPase, Suggesting Structural Flexibility at the Top of V1. International Journal of Molecular Sciences. 2011; 12(7):4693-4704.
Nowakowski, Szczepan; Mijaljica, Dalibor; Prescott, Mark; Devenish, Rodney J. 2011. "Vma8p-GFP Fusions Can Be Functionally Incorporated into V-ATPase, Suggesting Structural Flexibility at the Top of V1." Int. J. Mol. Sci. 12, no. 7: 4693-4704.