D. L. Murdza-Inglis, H. V. Patel, K. B. Freeman, P. Jezek, D. E.
Orosz, and K. D. Garlid (1991) Functional reconstitution of rat uncoupling
protein following its high level expression in yeast. J. Biol. Chem. 266,
11871-11875.
Abstract:
Small mammals, including human infants, rely on nonshivering thermogenesis
for a substantial portion of their body heat during exposure to cold. This
thermogenesis is mediated in large part by the uncoupling protein, which is
found exclusively within the inner membrane of brown adipose tissue mitochondria.
The sole function of uncoupling protein is to provide a regulated transport
pathway for electrophoretic back-flux of H+ ions into the mitochondrial matrix,
thereby dissipating the protonmotive force and producing heat. Thus, uncoupling
protein is unique with respect to both its physiological role and its tissue
expression. We have now achieved high level expression of rat uncoupling protein
in yeast, with import into yeast mitochondria at levels, 70-100 micrograms/mg
of mitochondrial protein, similar to those observed in brown adipose tissue
mitochondria from cold-adapted rats. When the expressed protein was purified
and reconstituted into liposomes, the proteoliposomes exhibited GDP-sensitive
proton and chloride uniports that were inhibited by GDP with Ki values similar
to those obtained with native protein. Moreover, the molecular activities
of the expressed protein with respect to Cl- and H+ transport were indistinguishable
from those of native protein. The availability of unlimited amounts of functional,
expressed uncoupling protein will now permit application of site-directed
mutagenesis to the many intriguing aspects of uncoupling protein structure
and function.
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