Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system.
The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.
Chen
Kun
K
Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.
Sun
Guoxun
G
Lv
Zhiyuan
Z
Wang
Chen
C
Jiang
Xueyuan
X
Li
Donghai
D
Zhang
Chenyu
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043344:Chordata / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011522:Protons; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic; D000071246:Uncoupling Protein 2
D007473:Ion Channels; D024101:Mitochondrial Proteins; D011522:Protons; C000604389:UCP2 protein, human; C000604391:Ucp2 protein, rat; D000071246:Uncoupling Protein 2; D006153:Guanosine Diphosphate