Folding Status Is Determinant over Traffic-Competence in Defining CFTR Interactors in the Endoplasmic Reticulum.

João D Santos,Sara Canato,Rune Matthiesen,Andre O Falcao,Carlos M Farinha,Kerman Aloria,Hugo M Botelho,Margarida D Amaral,Ana S Carvalho

doi:10.3390/cells8040353

Abstract

The most common cystic fibrosis-causing mutation (F508del, present in ~85% of CF patients) leads to CFTR misfolding, which is recognized by the endoplasmic reticulum (ER) quality control (ERQC), resulting in ER retention and early degradation. It is known that CFTR exit from the ER is mediated by specific retention/sorting signals that include four arginine-framed tripeptide (AFT) retention motifs and a diacidic (DAD) exit code that controls the interaction with the COPII machinery. Here, we aim at obtaining a global view of the protein interactors that regulate CFTR exit from the ER. We used mass spectrometry-based interaction proteomics and bioinformatics analyses to identify and characterize proteins interacting with selected CFTR peptide motifs or full-length CFTR variants retained or bypassing these ERQC checkpoints. We conclude that these ERQC trafficking checkpoints rely on fundamental players in the secretory pathway, detecting key components of the protein folding machinery associated with the AFT recognition and of the trafficking machinery recognizing the diacidic code. Furthermore, a greater similarity in terms of interacting proteins is observed for variants sharing the same folding defect over those reaching the same cellular location, evidencing that folding status is dominant over ER escape in shaping the CFTR interactome.

Highlights

Cystic fibrosis (CF) is the most common lethal monogenic autosomal recessive disease among the Caucasian population, and it is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which normally functions as a chloride/bicarbonate channel at the apical plasma membrane (PM) of epithelial cells [1]
To expand previous knowledge on the ERQC for CFTR, we aimed at identifying potential new traffic factors that interact with CFTR at the endoplasmic reticulum (ER)
We generated a novel CFBE41o− cell line stably expressing the DD/AA-CFTR variant. This novel cell line was characterized in terms of protein expression and response to modulators (Figure S1), and results showed that it recapitulated the previous findings for DD/AA-CFTR expressed in BHK cells, namely: the absence of mature CFTR and the lack of response to VX-809

Summary

Introduction

Cystic fibrosis (CF) is the most common lethal monogenic autosomal recessive disease among the Caucasian population, and it is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which normally functions as a chloride/bicarbonate channel at the apical plasma membrane (PM) of epithelial cells [1]. F508del causes CFTR misfolding, which is recognized by the endoplasmic reticulum (ER) quality control (ERQC), resulting in ER retention and targeting for degradation by the ubiquitin proteasome pathway (UPP) [2]. Understanding the exact mechanism that retains misfolded F508del-CFTR in the ER has been a major focus in the CF field. Both the folding and trafficking machineries play a relevant role in regulating. ER retention and exit of CFTR [2,3], but it is not completely understood to what extent misprocessing of F508del-CFTR results from the ER retention and/or its failure in interacting with COPII export machinery. CFTR exit from the ER has been proposed to involve at least four different checkpoints, the first two of which are “folding” checkpoints, involving mainly interaction with molecular chaperones, and the last two are “trafficking” checkpoints involving recognition of trafficking/exit signals to allow its exit and incorporation into vesicles [3,4,5,6]

Methods

Results

Conclusion