Abstract
A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidine-tagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with beta-galactosidase, resulted in a dose-dependent shift in kinetics indicating increased H(+) flux at any given membrane potential. UCP1 also enhanced H(+) flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H(+) conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.
Highlights
The issue of whether uncoupling protein (UCP) homologues mediate the proton leak is uncertain for mammalian cells since studies of expressed UCP homologues have largely been carried out in yeast and proteoliposomes [3, 4]
We assessed the effect of adenoviral overexpression of UCP2 on the proton leak in mitochondria isolated from rat insulinoma (INS-1) cells, a mammalian cell line in which UCP2 is present as a native protein
Hϩ flux as a function of membrane potential was determined in mitochondria isolated from INS-1 cells infected with different viral titers of adenoviral UCP2 or -galactosidase (Fig. 1)
Summary
We assessed the effect of adenoviral overexpression of UCP2 on the proton leak in mitochondria isolated from rat insulinoma (INS-1) cells, a mammalian cell line in which UCP2 is present as a native protein. The proton leak was measured as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential under conditions wherein oxygen use is leak-dependent and proportional to Hϩ transport To our knowledge, these are the first cellular studies to use this approach for assessment of the effect of an overexpressed UCP homologue on leak-dependent Hϩ conductance in mammalian mitochondria. None of the above studies was controlled for possible nonspecific effects of overexpression of mitochondrial membrane protein per se It uncertain whether catalysis of the proton leak, within a given cell type, is a property restricted to the naturally expressed UCP(s) within that cell type. The issue of nonspecific effects was addressed through overexpression of another mitochondrial membrane carrier protein, the oxoglutarate malate carrier (OMC) [9]
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