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A 100 kDa polypeptide associates with the V o membrane sector but not with the active oat vacuolar H + ‐ATPase, suggesting a role in assembly
Author(s) -
Li Xuhang,
Sze Heven
Publication year - 1999
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1999.00345.x
Subject(s) - protein subunit , immunostaining , immunoprecipitation , atpase , biology , biochemistry , cytosol , microbiology and biotechnology , v atpase , enzyme , yeast , membrane protein , membrane , gene , immunohistochemistry , immunology
Summary 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. & Sze H. (1992)Plant Physiol. 99, 925–931). A 100 kDa subunit of the yeast V‐ATPase is encoded byVPH1. 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 V o 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 V 1 subcomplexes in the cytosol. The free V 1 subcomplex became attached to the detergent‐solubilized V o 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 V o subcomplex would support a role of this protein in V‐ATPase assembly and perhaps in sorting.