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 dissipates the proton electrochemical potential gradient as heat. UCP activity is regulated: purine nucleotides inhibit while fatty acids activate transport. We have previously reported that sequence 261-269 of the UCP has a closely related counterpart in the adenine nucleotide translocator, as well as in the DNA binding domain of the estrogen receptor. 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 homologous domain (UCPDelta9) produced a highly deleterious mutant that collapsed the mitochondrial membrane potential and halted yeast growth. Since under our growth conditions revertants appeared rapidly, it was not possible to characterize this mutant. In this article, we have designed conditions to isolate mitochondria containing significant amounts of the UCPDelta9 mutant protein. These mitochondria show no respiratory control and are insensitive to nucleotides. Investigation of the permeability properties revealed that UCPDelta9 mitochondria swell rapidly in potassium salts in the absence of valinomycin, thus indicating a loss of specificity. The size exclusion properties of this mutant were determined with polyethylene glycols of various molecular masses (400-20000 Da), and it was found that UCPDelta9 can catalyze permeation of molecules of up to 1000 Da. We conclude that the deletion of amino acids 261-269 converts the UCP into an unspecific pore.
González-Barroso
M M
MM
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, and CEREMOD, CNRS, Meudon, France.
Fleury
C
C
Levi-Meyrueis
C
C
Zaragoza
P
P
Bouillaud
F
F
Rial
E
E
D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D014475:Uncoupling Agents / Q000378:metabolism; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1