Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae.
The uncoupling protein (UCP) from mammalian brown adipose tissue is an integral component of the mitochondrial inner membrane where it dissipates the proton electrochemical gradient. UCP is transported into mitochondria from the cytosol but lacks a cleavable targeting peptide. We have expressed the rat UCP in Saccharomyces cerevisiae and shown that this protein, which is not normally found in yeast, is targeted to the mitochondria where it disrupts mitochondrial function, probably by uncoupling oxidative phosphorylation. The observed growth defect is dependent upon the level of expression of UCP. When the unmodified UCP cDNA is expressed in yeast under the control of the GAL10 promoter no defect in growth is observed. We have inserted the UCP coding sequence behind the strong phosphoglycerate kinase promoter under the control of the GAL1-10 upstream activation site and introduced a yeast consensus sequence (ATAATG) at the translation start site. We have found that UCP expressed in S. cerevisiae is targeted to mitochondria and that its expression induces a marked growth defect on non-fermentable carbon sources in a manner dependent on induction with galactose.
Bathgate
B
B
Institute of Cell and Molecular Biology, University of Edinburgh, UK.
Freebairn
E M
EM
Greenland
A J
AJ
Reid
G A
GA
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002244:Carbon / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D005285:Fermentation; D007473:Ion Channels; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1; D002244:Carbon; D004247:DNA