DELETION OF AMINO-ACIDS-261-269 IN THE BROWN FAT UNCOUPLING PROTEIN CONVERTS THE CARRIER INTO A PORE

The uncoupling protein (UCP) from brown adipose tissue mitochondria is a carrier that catalyzes proton re-entry into the matrix and thus dis sipates the proton electrochemical potential gradient as heat. UCP act ivity is regulated: purine nucleotides inhibit while fatty acids activ ate transport. We have previously reported that sequence 261-269 of th e UCP has a closely related counterpart in the adenine nucleotide tran slocator, as well as in the DNA binding domain of the estrogen recepto r, Site-directed mutagenesis of the UCP showed that deletion of amino acids 267-269 in the UCP abolished nucleotide inhibition [Bouillaud, F ., et al. (1994) EMBO J. 13, 1990-1997], Complete deletion of the homo logous domain (UCP Delta 9) produced a highly deleterious mutant that collapsed the mitochondrial membrane potential and halted yeast growth . Since under our growth conditions revertants appeared rapidly, it wa s not possible to characterize this mutant, In this article, we have d esigned conditions to isolate mitochondria containing significant amou nts of the UCP Delta 9 mutant protein. These mitochondria show no resp iratory control and are insensitive to nucleotides. Investigation of t he permeability properties revealed that UCP Delta 9 mitochondria swel l rapidly in potassium salts in the absence of valinomycin, thus indic ating a loss of specificity. The size exclusion properties of this mut ant were determined with polyethylene glycols of various molecular mas ses (400-20000 Da), and it was found that UCP Delta 9 can catalyze per meation of molecules of up to 1000 Da, We conclude that the deletion o f amino acids 261-269 converts the UCP into an unspecific pore.