TY - JOUR
T1 - An energy-dependent maturation step is required for release of the cystic fibrosis transmembrane conductance regulator from early endoplasmic reticulum biosynthetic machinery
AU - Oberdorf, Jon
AU - Pitonzo, David
AU - Skach, William R.
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M504200200
DO - 10.1074/jbc.M504200200
M3 - Article
C2 - 16166089
AN - SCOPUS:33644689424
SN - 0021-9258
VL - 280
SP - 38193
EP - 38202
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -