Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1

Szczepan Nowakowski, Dalibor Mijaljica, Mark Prescott, Rodney Devenish

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

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 I?, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit I?, 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.
Original languageEnglish
Pages (from-to)4693 - 4704
Number of pages12
JournalInternational Journal of Molecular Sciences
Volume12
Issue number7
DOIs
Publication statusPublished - 2011

Cite this

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title = "Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1",
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 I?, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit I?, 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.",
author = "Szczepan Nowakowski and Dalibor Mijaljica and Mark Prescott and Rodney Devenish",
year = "2011",
doi = "10.3390/ijms12074693",
language = "English",
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pages = "4693 -- 4704",
journal = "International Journal of Molecular Sciences",
issn = "1422-0067",
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Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1. / Nowakowski, Szczepan; Mijaljica, Dalibor; Prescott, Mark; Devenish, Rodney.

In: International Journal of Molecular Sciences, Vol. 12, No. 7, 2011, p. 4693 - 4704.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1

AU - Nowakowski, Szczepan

AU - Mijaljica, Dalibor

AU - Prescott, Mark

AU - Devenish, Rodney

PY - 2011

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N2 - 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 I?, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit I?, 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.

AB - 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 I?, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit I?, 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.

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