Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system

The human uncoupling protein 3 gene generates two mRNA transcripts, uncoupl ing protein 3L and uncoupling protein 3S, which are predicted to encode lon g and short forms of the uncoupling protein 3 protein, respectively. While uncoupling protein 3L is similar in length to the other known uncoupling pr oteins, uncoupling protein 3S lacks the last 37 C-terminal residues. A spli ce site mutation in the human uncoupling protein 3 gene, resulting in the e xclusive expression of uncoupling protein 3S, and a number of point mutatio ns in the uncoupling protein 3 gene have been described. This study compare s the biochemical activity of uncoupling protein 3S as well as three mutant s of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupl ing protein 3L utilizing a yeast expression system. All proteins were expre ssed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function. Both uncoupling protein 3S and uncoupli ng protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O-2 consumption by 26 and 48%, respectively, and decrease d the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodi de. In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L, caused a similar (33 and 35%, respectively) increase in state 4 respira tion, which was relatively small compared to uncoupling protein 1 (102% inc rease). A truncated version of uncoupling protein 3S, lacking the last thre e C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif t hat is highly conserved among all mitochondrial carriers, had a greatly red uced uncoupling activity. The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, mere similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O-2 consumption. The R282C m utant had a reduced effect compared to uncoupling protein 3L. In summary un coupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling p rotein 3L, although uncoupling protein 3S and the R282C mutant have a modes tly reduced function. (C) 1999 Federation of European Biochemical Societies .