Site-directed mutagenesis identifies residues in uncoupling protein (UCP1)involved in three different functions

Using site-specific mutagenesis, we have constructed several mutants of unc oupling protein (UCP1) from brown adipose tissue to investigate the functio n of acidic side chains at positions 27, 167, 209, and 210 in H+ and Cl- tr ansport as well as in nucleotide binding. The H+ transport activity was mea sured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their Hf transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which s hows strong inhibition of H+ transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjac ent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H+ transport 80% and only 20%, respectively. These mutants r etain full Cl- transport activity. The results indicate that D210 participa tes in H+ uptake at the cytosolic side and D27 in H+ translocation through the membrane. Differently, E167Q has lost Cl- transport activity but retain s the ability to transport H+. The separate inactivation of H+ and Cl- tran sport argues against the fatty acid anion transport mechanism of H+ transpo rt by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not dip hosphate (NDP) binding. The results identify D209 and D210 in accordance wi th the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but nut of NDP binding.