An Energy-dependent Maturation Step Is Required for Release of the Cystic Fibrosis Transmembrane Conductance Regulator from Early Endoplasmic Reticulum Biosynthetic Machinery

Abstract

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.

Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

“…The TMD recruits calnexin to assist the polypeptide folding into the native conformation after its release from the translocon channel. Other proteins belonging to this category could be the viral fusion proteins of the paramyxovirus and rabies virus, which adopt different conformations in the absence of the TMD (23,24), and the cystic fibrosis transmembrane receptor whose TMDs are released from the translocon depending on the native folding of the ectodomain (25).…”

FKBP38 Peptidylprolyl Isomerase Promotes the Folding of Cystic Fibrosis Transmembrane Conductance Regulator in the Endoplasmic Reticulum

“…However, given that molecular chaperones have significant impact on multiple aspects of ion channel biogenesis including co-translational folding, post-translational maturation, ER quality control, and ER-associated degradation (ERAD) (13,14,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), before such a hypothesis can be tested directly a careful characterization of the FKBP38 role in membrane protein biogenesis needs to be conducted, and its functional relationship with Hsp90 must be defined. To this end we performed a systematic functional dissection of FKBP38 in the biogenesis of CFTR.…”

Cooperative Assembly and Misfolding of CFTR Domains In Vivo

“…The regulatory (R) domain is largely unstructured (Ostedgaard et al, 2000a), and, in coordination with the NBDs, regulates the gating of CFTR (Riordan, 2005). After cotranslational insertion into the ER, the nascent CFTR chain undergoes inefficient conformational maturation, mediated by molecular chaperones that require cytoplasmic ATP (Lukacs et al, 1994;Meacham et al, 1999;Oberdorf et al, 2005). Multiple quality control checkpoints ensure that native channels enter the secretory pathway via COPII transport vesicles (Wang et al, 2004;Wang et al, 2006;Younger et al, 2006), whereas partially folded molecules are eliminated by the endoplasmic reticulum (ER)-associated degradation (ERAD), using the ubiquitin-proteasome system (Kopito, 1999;Nakatsukasa and Brodsky, 2008).…”