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The 40-kDa subunit enhances but is not required for activity of the Coated vesicle proton pump

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K Puopolo
K Puopolo
K Puopolo
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M Sczekan
M Sczekan
M Sczekan
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R Magner
R Magner
R Magner
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Michael Forgac at Tufts University
Michael Forgac
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Abstract

We have previously demonstrated reassembly of a functional vacuolar (H+)-ATPase from clathrin-coated vesicles using the dissociated peripheral domain (V1) and the membrane-bound integral domain (V0) (Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836-14841). We have used this reassembly procedure to test the function of the 40-kDa subunit of the coated vesicle (H+)-ATPase. In the absence of V0, a fraction of the peripheral subunits reassemble into a V1 subcomplex which contains the 73-kDa A subunit, the 58-kDa B subunit, and the 34- and 33-kDa subunits but lacks the 40-kDa subunit. This subcomplex, which sediments with a mass of approximately 500 kDa, can be separated from the remaining monomeric subunits (and the 40-kDa subunit) by density gradient sedimentation. When dissociated with 0.36 M KI, 2.5 mM ATP, and 2.5 mM MgSO4, and added to membranes from which V1 has been dissociated, this V1(-40 kDa) subcomplex is able to reassemble with V0 to give a (H+)-ATPase with a proton pumping activity approximately half that obtained in the presence of the 40-kDa subunit. The undissociated subcomplex is not competent for assembly of a functional (H+)-ATPase. Interestingly, the monomeric fraction obtained from density gradient sedimentation contains the 40-kDa subunit but lacks the 34-kDa subunit. This monomeric fraction is nevertheless also able to assemble with V0 to give a functional proton pump. The V1V0 complexes assembled in the absence of either the 40- or 34-kDa subunits, while active, are not stable to detergent solubilization and immunoprecipitation, suggesting that both of these subunits play a role in stabilization of the (H+)-ATPase complex. Evidence for interaction between the 40- and 33-kDa subunits is also presented.
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... enhances, but is not essential for, v-ATPase function. 13 The relationship between those observations and our own will require additional study. ...
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Article
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Article
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  • Feb 1989
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Article
Full-text available
  • Jun 1988
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Full-text available
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Article
Full-text available
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Article
Full-text available
  • Jan 1992
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Article
Full-text available
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Article
Full-text available
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Functional reassembly of the coated vesicle proton pump
Article
Full-text available
  • Oct 1990
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Molecular cloning of cDNA encoding the C subunit of H+-ATPase from bovine chromaffin granules
Article
Full-text available
  • Dec 1990
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Ion motive ATPases. I. Ubiquity, properties, and significance to cell function
Article
  • Dec 1987
  • TRENDS BIOCHEM SCI
ATPases involved in ion translocation are present in a diverse variety of biological membranes. Three major classes of these ‘ion motive’ enzymes, designated here as ‘P’ (phosphorylated), ‘V’ (vacuolar), and ‘F’ (F0F1), are now known to exist. In addition to the regulation of intracellular ionic or pH balance, some ATPases help drive numerous physiological or biological processes by ‘energy coupling’. Among these are muscle relaxation, receptor recycling, hormone storage, food digestion and ATP synthesis. A number of laboratories are now trying to identify individual steps of ATPase reaction pathways, while others are endeavouring to identify those ‘genetic codes’ responsible for the energy coupling events.
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