Endoplasmic Reticulum Retention Motif Research Articles

Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABAB Receptors

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABABRs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABABRs are obligatory heteromers constituted by two subunits, GABABR1 and GABABR2. GABABR1a and GABABR1b are the most abundant subunit variants. GABABR1b is located in the somatodendritic domain whereas GABABR1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABABR1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABABRs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABABR1a operate in a pre-Golgi compartment. In the axon GABABR1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABABR1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABABRs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.

C-terminal Mutations Destabilize SIL1/BAP and Can Cause Marinesco-Sjögren Syndrome

Marinesco-Sjögren syndrome (MSS) is an autosomal recessive, neurodegenerative, multisystem disorder characterized by severe phenotypes developing in infancy. Recently, mutations in the endoplasmic reticulum (ER)-associated co-chaperone SIL1/BAP were identified to be the major cause of MSS. SIL1 acts as a nucleotide exchange factor for BiP, the ER Hsp70 orthologue, which plays an essential role in the folding and assembly of nascent polypeptide chains in the ER. SIL1 facilitates the release of BiP from unfolded protein substrates, enabling the subsequent folding and transport of the protein. Although most mutations leading to MSS result in deletion of the majority of the protein, three separate mutations have been identified that disrupt only the last five or six amino acids of the protein, which were assumed to encode a divergent ER retention motif. This study presents an in depth analysis of two of these mutants and reveals that the phenotype in the affected individuals is not likely to be due to depletion of SIL1 from the ER via secretion. Instead, our analyses show that the mutant proteins are particularly unstable and either form large aggregates in the ER or are rapidly degraded via the proteasome. In agreement with our findings, homology modeling suggests that the very C-terminal residues of SIL1 play a role in its structural integrity rather than its localization. These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein.

Dileucine and PDZ-binding Motifs Mediate Synaptic Adhesion-like Molecule 1 (SALM1) Trafficking in Hippocampal Neurons

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.

Optimization of Expression Conditions for Production of Anti-colorectal Cancer Monoclonal Antibody CO17-1A in Baculovirus-insect Cell System

The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 μL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 μL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.

Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking

The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.

Molecular cloning and characterization of the porcine Ero1L and ERp44 genes: Potential roles in controlling energy metabolism

Disulfide bond formation is a pivotal step in the maturation and release of secretory proteins that is controlled by specific endoplasmic reticulum (ER) resident enzymes. An important element in this process is Ero (ER oxidoreduction), a glycosylated flavoenzyme tightly associated with oxidative protein folding that lacks the known ER retention motifs. ER resident protein 44 kDa (ERp44) is an ER resident protein that mediates ERo1 localization in ER and also prevents the secretion of unassembled cargo proteins with unpaired cysteine. These proteins are not only the key participants in the disulfide-bond formation process, but they also control the secretory pathway on both qualitative and quantitative levels. Here, we cloned full-length cDNA sequences of the porcine Ero1L (1448 bp) and ERp44 (1361 bp) genes. Isolation and characterization of their genomic sequences revealed that Ero1L contains 16 exons and 15 introns almost 150 kp in length, whereas ERp44 contains 12 exons and 11 introns more than 140 kp in length, and they are located on porcine chromosome 1q21 and 1q29, respectively. Tissue distribution analysis of the two genes revealed extremely high expression in adipose tissue, and the topology of their phylogenic tree indicates a high degree of conservation among different species. We looked at transcription factors binding sites in the 5′-flanking regions of Ero1L and ERp44, and many adipose differentiations related factors reflect the tight relationship to energy metabolism.

Synthesis, Maturation, and Trafficking of Human Na+-Dicarboxylate Cotransporter NaDC1 Requires the Chaperone Activity of Cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Interaction of Autographa californica Multiple Nucleopolyhedrovirus Cathepsin Protease Progenitor (proV-CATH) with Insect Baculovirus Chitinase as a Mechanism for proV-CATH Cellular Retention

The insect baculovirus chitinase (CHIA) and cathepsin protease (V-CATH) enzymes cause terminal host insect liquefaction, enhancing the dissemination of progeny virions away from the host cadavers. Regulated and delayed cellular release of these host tissue-degrading enzymes ensures that liquefaction starts only after optimal viral replication has occurred. Baculoviral CHIA remains intracellular due to its C-terminal KDEL endoplasmic reticulum (ER) retention motif. However, the mechanism for cellular retention of the inactive V-CATH progenitor (proV-CATH) has not yet been determined. Signal peptide cleavage occurs upon cotranslational ER import of the v-cath-expressed protein, and ER-resident CHIA is needed for the folding of proV-CATH. Although this implies that CHIA and proV-CATH bind each other in the ER, the putative CHIA-proV-CATH interaction has not been experimentally verified. We demonstrate that the amino-terminal 22 amino acids (aa) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) preproV-CATH are responsible for the entry of proV-CATH into the ER. Furthermore, the CHIA-green fluorescent protein (GFP) and proV-CATH-red fluorescent protein (RFP) fusion proteins colocalize in the ER. Using monomeric RFP (mRFP)-based bimolecular fluorescence complementation (BiFC), we determined that CHIA and proV-CATH interact directly with each other in the ER during virus replication. Moreover, reciprocal Ni/His pulldowns of His-tagged proteins confirmed the CHIA-proV-CATH interaction biochemically. The reciprocal copurification of CHIA and proV-CATH suggests a specific CHIA-proV-CATH interaction and corroborates our BiFC data. Deletion of the CHIA KDEL motif allowed for premature CHIA secretion from cells, and proV-CATH was similarly prematurely secreted from cells along with ΔKDEL-CHIA. These data suggest that CHIA and proV-CATH interact directly with each other and that this interaction aids the cellular retention of proV-CATH.

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi

Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L 6E 9 myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.

Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP

The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr(648) of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented.

An Arginine Stretch Limits ADAM10 Exit from the Endoplasmic Reticulum

A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich ((723)RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10.

Human invariant chain Iip35 isoform expressed in Ii-deficient mice fully restores thymic selection (130.16)

Abstract During biosynthesis, invariant chain (Ii) associates with MHC II molecules, prevents peptide binding in the endoplasmic reticulum (ER) and targets the complexes to the endocytic pathway. Iip35, a human-specific isoform, displays a cytoplasmic ER retention motif that is masked upon MHC II binding. Little is known on the role of this ER retention motif and we asked if Iip35 alone was capable of supporting the functions of Ii. We generated a Tg mouse expressing the human Iip35 isoform (Tgp35). Breeding with murine Ii deficient mice produced mice expressing only Iip35 (Tgp35/mIiKO). In these mice, Iip35 is highly expressed by about 30 % of B cells, 50% of splenic DC and 74% of IFNγ-induced macrophages. Interestingly, B cells expressing Iip35 were lost during the maturation process and not found in the T2 stage. We have demonstrated that Iip35 interacts with both murine Ii and MHC II. Also, we observed MHC II compacts forms typical of tight peptide binding. Iip35 partially rescued MHC II cell surface expression in mIiKO mice and was sufficient to fully restore thymic selection as judged by normal CD4+ T cell numbers in thymus and periphery. In contrast, as observed in mIiKO mice, no antigen-specific antibody production was observed in Tgp35/mIiKO following injection of Ova. Altogether, our results suggest that the Iip35 isoform is able to restore thymic selection but may not be sufficient to perform all Ii functions.

Molecular cloning and functional characterization of porcine stimulator of interferon genes (STING)

The stimulator of interferon genes (STING) of human/mouse has been identified recently as an adaptor that links virus-sensing receptors to interferon regulatory factor 3 (IRF3) activation. Here we report the cloning and characterization of porcine STING (poSTING). The full-length poSTING cDNA sequence encodes 378 amino acids and contains one endoplasmic reticulum (ER) retention motif, RAR. Phylogenetic analysis revealed that poSTING, together with bovine STING, is more closely related to the human than to mouse STING. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Enhanced green fluorescence protein (EGFP)-labeled poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and nuclear factor κB (NF-κB) transcription factors to induce IFN-β production, while knockdown of poSTING significantly inhibited poly(I:C)- and poly(dAT:dAT)-induced IFN-β promoter activation and IFN-β mRNA production. Furthermore, the pseudorabies virus (PRV), a dsDNA virus, has been shown to activate the IFN-β promoter in a poSTING-dependent way in porcine cell lines. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution.

BLAP-Tagged Channel Technology: A New Direction Trafficking toward Epithelial Physiology

BLAP-Tagged Channel Technology: A New Direction Trafficking toward Epithelial Physiology

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel–Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Expression of fully assembled TCR-CD3 complex on double positive thymocytes: synergistic role for the PRS and ER retention motifs in the intra-cytoplasmic tail of CD3

TCR expression on double-positive (DP) thymocytes is a prerequisite for thymic selection that results in the generation of mature CD4(+) and CD8(+) single-positive T cells. TCR is expressed at very low level on preselection DP thymocytes and is dramatically up-regulated on positively selected thymocytes. However, mechanism governing TCR expression on developing thymocytes is not understood. In the present report, we demonstrate that the intra-cytoplasmic (IC) domain of CD3epsilon plays a critical role in regulating TCR expression on DP thymocytes. We provide genetic and biochemical evidence to show that the CD3epsilon IC domain mutations result in elevated expression of fully assembled TCR on DP thymocytes. We also demonstrate that TCR up-regulation on DP thymocytes in these transgenic mice occurs in a ligand-independent manner. Further, we show that the proline-rich sequence and endoplasmic reticulum (ER) retention motifs in the IC domain of CD3epsilon play synergistic role in regulating TCR surface expression on DP thymocytes.

20th National Meeting of the British Neuroscience Association

20th National Meeting of the British Neuroscience Association

A Role for SNAP25 in Internalization of Kainate Receptors and Synaptic Plasticity

Regulation of surface insertion and internalization of AMPA and NMDA receptors has emerged as a key mechanism for the control of synaptic strength. Regulatory elements for synaptic kainate receptors (KARs) are, however, largely undetermined. We have found that SNAP25 is critical for the synaptic removal of KARs, acting via GluK5 (i.e., KA2) subunits. SNAP25 coimmunoprecipitates with protein complexes containing PICK1, GRIP1, and GluK5 and colocalizes with GluK5 in both hippocampal neurons and transfected HEK293 cells. In hippocampal slices, purified SNAP25 antibodies and blocking peptides caused a GluK5-dependent run-up of KARs-mediated EPSC (EPSC(KAR)) recorded from CA3 pyramidal neurons when included in the patch pipette and prevented activity-dependent long-term depression of EPSC(KAR). As EPSC(KAR) LTD, SNAP25/PICK1/GluK5 interactions are dynamically regulated by PKC.

Functional Rescue of DeltaF508-CFTR by Peptides Designed to Mimic Sorting Motifs

The cystic fibrosis (CF)-causing mutant, deltaF508-CFTR, is misfolded and fails to traffic out of the endoplasmic reticulum (ER) to the cell surface. Introduction of second site mutations that disrupt a diarginine (RXR)-based ER retention motif in the first nucleotide binding domain rescues the trafficking defect of deltaF508-CFTR, supporting a role for these motifs in mediating ER retention of the major mutant. To determine if these RXR motifs mediate retention of the native deltaF508-CFTR protein in situ, we generated peptides that mimic these motifs and should antagonize mistrafficking mediated via their aberrant exposure. Here we show robust rescue of deltaF508-CFTR in cell lines and in respiratory epithelial tissues by transduction of RXR motif-mimetics, showing that abnormal accessibility of this motif is a key determinant of mistrafficking of the major CF-causing mutant.