Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis

X. S. Xie, D. K. Stone

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S. Z., Xie, X. S., and Stone D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase- and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3 -, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

Original languageEnglish (US)
Pages (from-to)9859-9867
Number of pages9
JournalJournal of Biological Chemistry
Volume263
Issue number20
StatePublished - 1988

Fingerprint

Clathrin-Coated Vesicles
Clathrin
Calcium-Transporting ATPases
Protons
Hydrolysis
Adenosine Triphosphate
Adenosine Triphosphatases
Ca(2+) Mg(2+)-ATPase
Proton Pumps
Holoenzymes
Peptides
Ethylmaleimide
Centrifugation
Phosphatidylserines
Glycerol
Genetic Recombination
Urea
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{eadfc654e07844389e50df7720ad2192,
title = "Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis",
abstract = "The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S. Z., Xie, X. S., and Stone D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase- and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3 -, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.",
author = "Xie, {X. S.} and Stone, {D. K.}",
year = "1988",
language = "English (US)",
volume = "263",
pages = "9859--9867",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis

AU - Xie, X. S.

AU - Stone, D. K.

PY - 1988

Y1 - 1988

N2 - The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S. Z., Xie, X. S., and Stone D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase- and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3 -, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

AB - The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S. Z., Xie, X. S., and Stone D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase- and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3 -, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

UR - http://www.scopus.com/inward/record.url?scp=0023792760&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023792760&partnerID=8YFLogxK

M3 - Article

C2 - 2454921

AN - SCOPUS:0023792760

VL - 263

SP - 9859

EP - 9867

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -