A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H+-ATPase, suggesting a role in assembly

Xuhang Li, Heven Sze

Research output: Contribution to journalArticle

Abstract

The vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells. Although a 100 kDa subunit is common to many V-ATPases, it is not detected in a purified and active pump from oat (Ward J.M. and Sze H. (1992) Plant Physiol. 99, 925-931). A 100 kDa subunit of the yeast V-ATPase is encoded by VPH1. Immunostaining revealed a Vph1p-related polypeptide in oat membranes, thus the 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 an approximately 250 kDa V0 complex including subunits of 36 and 16 kDa. Immunostaining with an affinity-purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the ≃ 670 kDa holoenzyme. Co-immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol. The free V1 subcomplex became attached to the detergent-solubilized V0 sector after mixing, as subunits of both sectors were co-precipitated by an antibody against subunit A. The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity. Its association with the free V0 subcomplex would support a role of this protein in V-ATPase assembly and perhaps in sorting.

Original languageEnglish (US)
Pages (from-to)19-30
Number of pages12
JournalPlant Journal
Volume17
Issue number1
DOIs
StatePublished - Jan 1999

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Vacuolar Proton-Translocating ATPases
H-transporting ATP synthase
oats
polypeptides
Peptides
Membranes
detergents
Detergents
Adenosine Triphosphatases
Yeasts
Free Association
yeasts
Holoenzymes
Antibody Affinity
Eukaryotic Cells
Immunoprecipitation
cytosol
membrane proteins
pumps
Cytosol

ASJC Scopus subject areas

  • Plant Science

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A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H+-ATPase, suggesting a role in assembly. / Li, Xuhang; Sze, Heven.

In: Plant Journal, Vol. 17, No. 1, 01.1999, p. 19-30.

Research output: Contribution to journalArticle

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