Herve Barriere

Peripheral Protein Quality Control Removes Unfolded CFTR from the Plasma Membrane

Peripheral Quality Control Protein misfolding diseases often lead to the retention and degradation of important proteins within the endoplasmic reticulum (ER). Strategies to reduce the stringency of ER quality control that allow the proteins to carry on through the secretory pathway to reach their destination at the cell surface have shown some promise. Okiyoneda et al. (p. 805, published online 1 July; see the Perspective by Hutt and Balch) wanted to understand how, even if a protein reaches its destination, it may still be subjected to a second level of quality control and be cleared from the plasma membrane. Using functional small-interfering RNA screens in cells expressing the common cystic fibrosis mutation F508CFTR, the authors identified a pair of chaperones that promoted clearance of defective proteins from the plasma membrane. This peripheral quality-control step will also need to be overcome to increase the effectiveness of strategies to overcome protein misfolding disorders. Cells clear misfolded and damaged proteins from the cell surface, sometimes frustrating attempts to treat protein-folding diseases. Therapeutic efforts to restore biosynthetic processing of the cystic fibrosis transmembrane conductance regulator lacking the F508 residue (ΔF508CFTR) are hampered by ubiquitin-dependent lysosomal degradation of nonnative, rescued ΔF508CFTR from the plasma membrane. Here, functional small interfering RNA screens revealed the contribution of chaperones, cochaperones, and ubiquitin-conjugating and -ligating enzymes to the elimination of unfolded CFTR from the cell surface, as part of a peripheral protein quality-control system. Ubiquitination of nonnative CFTR was required for efficient internalization and lysosomal degradation. This peripheral protein quality-control mechanism probably participates in the preservation of cellular homeostasis by degrading damaged plasma membrane proteins that have escaped from the endoplasmic reticulum quality control or are generated by environmental stresses in situ.

Molecular Basis of Oligoubiquitin‐Dependent Internalization of Membrane Proteins in Mammalian Cells

Ubiquitination induced down‐regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub‐interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub‐binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin‐dependent, but caveolin‐independent. Functional assays demonstrate the cargo‐dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated β2‐adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin‐mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM–Ub interactions to overcome the low affinity binding of mono‐Ub to UIM.

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway†

ABSTRACT The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.

Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr(+/+) and cftr(-/-) mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase-generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.

Ubiquitination-dependent quality control of hERG K+ channel with acquired and inherited conformational defect at the plasma membrane

The role of the plasma membrane quality control machinery is demonstrated in the development of the long QT syndrome phenotype, caused by acquired and inherited conformational defects of the hERG potassium channel in multiple expression systems, including cardiac myocytes.

Analysis of Endocytic Trafficking by Single‐Cell Fluorescence Ratio Imaging

The post‐endocytic sorting of internalized membrane proteins plays a critical role in numerous physiological processes, including receptor desensitization, degradation of non‐native plasma membrane proteins, and cell surface retrieval of receptors from early endosomes upon ligand dissociation. Here, we describe a fluorescence ratiometric image analysis (FRIA) method used to determine the post‐endocytic fate and transport kinetics of transmembrane proteins based on the pH measurement of internalized cargo‐containing compartments in living cells. The method relies on the notion that the pH of a cargo‐containing transport vesicle (vesicular pH, pHv) could be taken as an indicator of its identity, considering that endocytic organelles (e.g., sorting endosome, recycling endosome, late endosome/MVB, and lysosome) have characteristic pHv. The pH‐sensitive FITC‐conjugated secondary antibody is attached to the cargo via a primary antibody, recognizing the cargo extracellular domain. The pHv is determined by single‐cell FRIA. Internalized cargo colocalization with organellar markers, as well as pHv measurement of recycling endosome, lysosome, and the TGN are discussed to validate the technique and facilitate data interpretation. Curr. Protoc. Cell Biol. 40:15.13.1‐15.13.21.

Endocytic Sorting of CFTR Variants Monitored by Single-Cell Fluorescence Ratiometric Image Analysis (FRIA) in Living Cells

The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr- and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued F508del and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratiometric image analysis (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs.