Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex.

S Z Sun,X S Xie,D K Stone

doi:10.1016/s0021-9258(18)47864-7

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 mumol 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.

Highlights

Clathrin-coated vesicle acidification is mediatedby an elec- of this new class of proton pumps
The actual subunit trogenic proton translocating ATPase which is resistant to composition of these pumpsis not known and thepolypeptide oligomycin and efrapeptin, but is inhibbityedN-ethylmaleim- components of the isolates range from thrfeoer plant [13, 16]
The coated vesicle proton pump is inhibited vesicle complex [11].It is possible that subunits critical for by dicyclohexylcarbodiimide (DCCD)’ a t a DCCD/protein proton pumping are missing from preparations with apparratio over 100-fold greater than that required to inhibit the ently fewer components

Summary

RESULTS

The mixture was frozen at -70 "C for 15 min, the proteoliposomes were thawed on ice and thenwere added to 1.5 mlof assay buffer (described below) for measurement of ATP-generated acridine orange quenching. The reaction solution (1 ml) consisted of150 mM KC1 and 3 p~ presence of 5 pl of phosphatidylserine (0.5 mg/ml) and 0.5 p1 of valinomycin and calibration was performed afterexperimentsby DCCD solutions of varying concentrations which yielded the final injecting known quantities of HCl into the cuvette, designated concentration in the 15.5~1mixture. Samples were preincubated with 2.5pgof phosphatidylserine for 5 min at 25 "C in the presence of 0.5 p1of DCCD solutions of varying min at 25 'C, and thereaction was initiated by the addition of 200pl concentrations which yielded the final designated concentration in of assay solution and after a 10-min incubation at 37 "C, was termi- the 10.5-pl mixture. 3zPiwas assayed as described [21]

Proton Channel Isolation and Reconstitution

ONOFF ON OFF

ATPase preparation shares nopolypeptides in common with