An energy-dependent maturation step is required for release of the cystic fibrosis transmembrane conductance regulator from early endoplasmic reticulum biosynthetic machinery.
Polytopic proteins are synthesized in the endoplasmic reticulum (ER) by ribosomes docked at the Sec61 translocation channel. It is generally assumed that, upon termination of translation, polypeptides are spontaneously released into the ER membrane where final stages of folding and assembly are completed. Here we investigate early interactions between the ribosome-translocon complex and cystic fibrosis transmembrane conductance regulator (CFTR), a multidomain ABC transporter, and demonstrate that this is not always the case. Using in vitro and Xenopus oocyte expression systems we show that, during and immediately following synthesis, nascent CFTR polypeptides associate with large, heterogeneous, and dynamic protein complexes. Partial-length precursors were quantitatively isolated in a non-covalent, puromycin-sensitive complex (>3,500 kDa) that contained the Sec61 ER translocation machinery and the cytosolic chaperone Hsc70. Following the completion of synthesis, CFTR was gradually released into a smaller (600-800 kDa) ATP-sensitive complex. Surprisingly, release of full-length CFTR from the ribosome and translocon was significantly delayed after translation was completed. Moreover, this step required both nucleotide triphosphates and cytosol. Release of control proteins varied depending on their size and domain complexity. These studies thus identify a novel energy-dependent step early in the CFTR maturation pathway that is required to disengage nascent CFTR from ER biosynthetic machinery. We propose that, contrary to current models, the final stage of membrane integration is a regulated process that can be influenced by the state of nascent chain folding, and we speculate that this step is influenced by the complex multidomain structure of CFTR.
Oberdorf
Jon
J
Department of Biochemistry and Moleculor Biology, Oregon Health & Sciences University, Portland, Oregon 97239, USA.
Pitonzo
David
D
Skach
William R
WR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism* / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005990:Glycerol / Q000737:chemistry; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D000069816:SEC Translocation Channels; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014982:Xenopus laevis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003432:Cross-Linking Reagents; D050883:HSC70 Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012313:RNA; D000255:Adenosine Triphosphate; D005990:Glycerol