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, uncoupling protein 3L and uncoupling protein 3S, which are predicted to encode long and short forms of the uncoupling protein 3 protein, respectively. While uncoupling protein 3L is similar in length to the other known uncoupling proteins, uncoupling protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human uncoupling protein 3 gene, resulting in the exclusive expression of uncoupling protein 3S, and a number of point mutations in the uncoupling protein 3 gene have been described. This study compares the biochemical activity of uncoupling protein 3S as well as three mutants of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupling protein 3L utilizing a yeast expression system. All proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function. Both uncoupling protein 3S and uncoupling protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to uncoupling protein 1 (102% increase). A truncated version of uncoupling protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced uncoupling activity. The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, were similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption. The R282C mutant had a reduced effect compared to uncoupling protein 3L. In summary, uncoupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling protein 3L, although uncoupling protein 3S and the R282C mutant have a modestly reduced function.