A 100 kDa polypeptide associates with the VO sector but not with the active oat vacuolar H+-ATPase suggesting a role in assembly

Xuhang Li, Sze Heven

Research output: Contribution to journalArticle

Abstract

Vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembranc compartments in eukaryotic cells. Although a 100-kDa subunit is common to many V-ATPases, it is absent from a purified and active pump of oat. Im munostaining revealed a Vphlp-related polypeptide in oat membranes, thus tho role of this polypeptide was investigated. Membrane proteins were detergent solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100-kDa polypeptide was not associated with the fully assembled ATPase; however it was part of a 250 kDa V0 complex including subunits of 36- and 16-kDa. coimmunoprecipitation with a monoclonal anti body against A subunit indicated that free Vi subcomplexes existed in the cytosol. The free Vi subcomplex became attached to the detergent-solubilized V0 sector after mixing, and an antibody against subunit A coprecipitated all Vj and Yû subunits except for the 100-kDa polypeptide. The absence of this polypeptide from the active enzyme demonstrates that, unlike the yeast Vphlp, the 100-kDa polypeptide in oat is not required for activity. Its association with the free V0 sector at the endoplasmic reticulum would support a role of this protein in the assembly of the VD subcomplex and of the entire V-ATPase.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998
Externally publishedYes

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Vacuolar Proton-Translocating ATPases
H-transporting ATP synthase
oats
polypeptides
Peptides
detergents
Detergents
Adenosine Triphosphatases
Free Association
Eukaryotic Cells
cytosol
membrane proteins
Endoplasmic Reticulum
pumps
Cytosol
endoplasmic reticulum
Yeast
adenosinetriphosphatase
eukaryotic cells
Avena

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

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title = "A 100 kDa polypeptide associates with the VO sector but not with the active oat vacuolar H+-ATPase suggesting a role in assembly",
abstract = "Vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembranc compartments in eukaryotic cells. Although a 100-kDa subunit is common to many V-ATPases, it is absent from a purified and active pump of oat. Im munostaining revealed a Vphlp-related polypeptide in oat membranes, thus tho role of this polypeptide was investigated. Membrane proteins were detergent solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100-kDa polypeptide was not associated with the fully assembled ATPase; however it was part of a 250 kDa V0 complex including subunits of 36- and 16-kDa. coimmunoprecipitation with a monoclonal anti body against A subunit indicated that free Vi subcomplexes existed in the cytosol. The free Vi subcomplex became attached to the detergent-solubilized V0 sector after mixing, and an antibody against subunit A coprecipitated all Vj and Y{\^u} subunits except for the 100-kDa polypeptide. The absence of this polypeptide from the active enzyme demonstrates that, unlike the yeast Vphlp, the 100-kDa polypeptide in oat is not required for activity. Its association with the free V0 sector at the endoplasmic reticulum would support a role of this protein in the assembly of the VD subcomplex and of the entire V-ATPase.",
author = "Xuhang Li and Sze Heven",
year = "1998",
language = "English (US)",
volume = "12",
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TY - JOUR

T1 - A 100 kDa polypeptide associates with the VO sector but not with the active oat vacuolar H+-ATPase suggesting a role in assembly

AU - Li, Xuhang

AU - Heven, Sze

PY - 1998

Y1 - 1998

N2 - Vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembranc compartments in eukaryotic cells. Although a 100-kDa subunit is common to many V-ATPases, it is absent from a purified and active pump of oat. Im munostaining revealed a Vphlp-related polypeptide in oat membranes, thus tho role of this polypeptide was investigated. Membrane proteins were detergent solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100-kDa polypeptide was not associated with the fully assembled ATPase; however it was part of a 250 kDa V0 complex including subunits of 36- and 16-kDa. coimmunoprecipitation with a monoclonal anti body against A subunit indicated that free Vi subcomplexes existed in the cytosol. The free Vi subcomplex became attached to the detergent-solubilized V0 sector after mixing, and an antibody against subunit A coprecipitated all Vj and Yû subunits except for the 100-kDa polypeptide. The absence of this polypeptide from the active enzyme demonstrates that, unlike the yeast Vphlp, the 100-kDa polypeptide in oat is not required for activity. Its association with the free V0 sector at the endoplasmic reticulum would support a role of this protein in the assembly of the VD subcomplex and of the entire V-ATPase.

AB - Vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembranc compartments in eukaryotic cells. Although a 100-kDa subunit is common to many V-ATPases, it is absent from a purified and active pump of oat. Im munostaining revealed a Vphlp-related polypeptide in oat membranes, thus tho role of this polypeptide was investigated. Membrane proteins were detergent solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100-kDa polypeptide was not associated with the fully assembled ATPase; however it was part of a 250 kDa V0 complex including subunits of 36- and 16-kDa. coimmunoprecipitation with a monoclonal anti body against A subunit indicated that free Vi subcomplexes existed in the cytosol. The free Vi subcomplex became attached to the detergent-solubilized V0 sector after mixing, and an antibody against subunit A coprecipitated all Vj and Yû subunits except for the 100-kDa polypeptide. The absence of this polypeptide from the active enzyme demonstrates that, unlike the yeast Vphlp, the 100-kDa polypeptide in oat is not required for activity. Its association with the free V0 sector at the endoplasmic reticulum would support a role of this protein in the assembly of the VD subcomplex and of the entire V-ATPase.

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