ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop.
Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H(+) efflux with K(m) of 56.36+/-0.27microM and V(max) of 66.9micromolH(+)min(-1)(mgprot)(-1). LA-mediated H(+) fluxes were sensitive to ATP inhibition with K(i) of 2.61+/-0.36mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism.
Fávaro
Regiane Degan
RD
Instituto de Biociências, Departamento de Genética, UNESP, Botucatu, SP, Brazil.
Borecký
Jiri
J
Colombi
Débora
D
Maia
Ivan G
IG
D000255:Adenosine Triphosphate / Q000737:chemistry; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007473:Ion Channels; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D024101:Mitochondrial Proteins; D009154:Mutation; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000071256:Uncoupling Protein 1; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D018076:DNA, Complementary; D005227:Fatty Acids; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D011522:Protons; D011994:Recombinant Proteins; D000071256:Uncoupling Protein 1; D006639:Histidine; D000255:Adenosine Triphosphate; D019787:Linoleic Acid