The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins.
The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier. We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli. Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP. Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained. The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane. Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP. This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins.
Miroux
B
B
Centre de Recherches en Endocrinologie Moléculaire et Développement (CEREMOD), CNRS, Meudon, France.
Frossard
V
V
Raimbault
S
S
Ricquier
D
D
Bouillaud
F
F
D018528:ATP-Binding Cassette Transporters; D002001:Adipose Tissue, Brown / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015723:Gene Library; D007106:Immune Sera / Q000276:immunology; D007473:Ion Channels; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D008928:Mitochondria / Q000737:chemistry*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D000071256:Uncoupling Protein 1
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D007106:Immune Sera; D007473:Ion Channels; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D000071256:Uncoupling Protein 1; C461599:maltose transport system, E coli; D004247:DNA