Abstract
The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 μmol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 14790-14794 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 262 |
| Issue number | 30 |
| State | Published - 1987 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex. / Sun, S. Z.; Xie, X. S.; Stone, D. K.
In: Journal of Biological Chemistry, Vol. 262, No. 30, 1987, p. 14790-14794.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex
AU - Sun, S. Z.
AU - Xie, X. S.
AU - Stone, D. K.
PY - 1987
Y1 - 1987
N2 - The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 μmol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.
AB - The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 μmol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.
UR - http://www.scopus.com/inward/record.url?scp=0023204737&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023204737&partnerID=8YFLogxK
M3 - Article
VL - 262
SP - 14790
EP - 14794
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -