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Activity and in vitro reassembly of the coated vesicle (H+)-ATPase requires the 50-kDa subunit of the clathrin assembly complex AP-2.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1994 Dec 16; Vol. 269 (50), pp. 31592-7. - Publication Year :
- 1994
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Abstract
- We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.
- Subjects :
- Adaptor Proteins, Vesicular Transport
Animals
Brain
Cattle
Hydrogen-Ion Concentration
In Vitro Techniques
Macromolecular Substances
Structure-Activity Relationship
Adaptor Protein Complex 2
Adaptor Protein Complex mu Subunits
Coated Vesicles enzymology
Nerve Tissue Proteins metabolism
Phosphoproteins metabolism
Proton-Translocating ATPases metabolism
Vacuoles enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 269
- Issue :
- 50
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 7989329