Faculty Collaboration Database

Mutagenesis of the uncoupling protein of brown adipose tissue. Neutralization Of E190 largely abolishes pH control of nucleotide binding. Biochemistry 1997 Jul 08;36(27):8253-60

Date

07/08/1997

Pubmed ID

9204870

DOI

10.1021/bi970513r

Scopus ID

2-s2.0-0030836775 (requires institutional sign-in at Scopus site)   55 Citations

Abstract

For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein (UCP) of brown adipose tissue from hamster has been isolated and used to transform yeast cells. Optimized expression conditions yielded 2% of mitochondrial protein as UCP. UCP was isolated, avoiding copurification of ADP/ATP carrier and porin. Intrahelical E190, previously suggested to be the pH sensor for nucleotide binding, was neutralized to glutamine by mutagenesis. In binding titrations with [14C]guanosine 5'-triphosphate (GTP) and with fluorescent dansyl-GTP, near equal binding capacity for GTP was measured in wild-type (wt) and E190Q. The KD for GTP binding to UCP from yeast has the same strong pH dependence as the original UCP from hamster. With both [14C]GTP and dansyl-GTP, the KD in wt increased 16-19-fold from pH 6.0 to 7.5, while in E190Q this increase was only 2.5-2.9-fold. As a result, at pH 7.5, both [14C]GTP and dansyl-GTP bind 6-fold tighter to E190Q than to wt. The binding rate of GTP decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in E190Q. Woodward reagent K (WRK) known to interact specifically with E190 [Winkler, E., Wachter, E., and Klingenberg, M. (1997) Biochemistry 36, 148-155] abolished [14C]GTP and dansyl-GTP binding to wt UCP, whereas binding to E190Q was fully resistant to WRK. H+ and Cl- transport activity in reconstituted vesicles were the same with wt and E190Q. At pH 7.5, 5 microM GTP is unable to inhibit H+ and Cl- transport in wt but inhibits in E190Q to maximum level. The different sensitivity toward GTP versus GDP found in wt is absent in E190Q. Thus, the mutation E190Q results in the predicted gain of function in binding and proves the role of the intrahelical E190 as a pH sensor for nucleotide binding but excludes a role in transport.

Author List

Echtay KS, Bienengraeber M, Klingenberg M

MESH terms used to index this publication - Major topics in bold

Adipose Tissue, Brown
Animals
Binding Sites
Carrier Proteins
Cricetinae
Dansyl Compounds
Exons
Galactose
Gene Transfer Techniques
Guanosine Diphosphate
Guanosine Triphosphate
Hydrogen-Ion Concentration
Immunoblotting
Ion Channels
Kinetics
Membrane Proteins
Mitochondria
Mitochondrial Proteins
Mutagenesis, Site-Directed
Nucleotides
Saccharomyces cerevisiae
Structure-Activity Relationship
Uncoupling Protein 1