Topogenesis of mitochondrial inner membrane uncoupling protein. Rerouting transmembrane segments to the soluble matrix compartment.
Brown adipose tissue uncoupling protein (UCP), an integral polytopic protein of the mitochondrial inner membrane, is composed of at least six transmembrane segments whose net hydrophobic character derives from paired amphiphilic helices. The protein is synthesized in the cytoplasm as a polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Deletion mutagenesis and fusion protein constructions revealed the existence of at least two import signals: one lying between UCP precursor amino acids 13-105 and the other downstream of position 101. The former resulted in both targeting and membrane insertion of a fusion protein, whereas the latter targeted UCP 102-307 into the organelle but failed to result in membrane insertion. When a strong matrix-targeting signal derived from precarbamoyl phosphate synthetase was fused to UCP amino acids 169-307 or 52-307 (containing three and five transmembrane domains, respectively), the fusion proteins were efficiently imported to the soluble matrix compartment where correct signal cleavage took place. We suggest that assembly of UCP into the inner membrane follows a coordinate insertion pathway for integration and may use more than one signal sequence to achieve this. In this respect, it might share certain mechanistic features with the insertion of polytopic proteins into the endoplasmic reticulum. The data also suggest, however, that integration of the amino-terminal third of UCP into the inner membrane may be required to help or enhance insertion of the remaining UCP transmembrane domains.
Department of Biochemistry, McGill University, Montreal, Canada.
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Liu
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Bell
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Freeman
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Shore
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D024101:Mitochondrial Proteins; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D051381:Rats; D014158:Transcription, Genetic; D014475:Uncoupling Agents / Q000235:genetics / 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; D004247:DNA