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Highlights • Co-translational folding of NBD1 is disrupted by CF mutations. • FRET analysis of transmembrane constructs reveals misfolding by CF mutations. • The impact of the F508del mutation on CFTR is species-dependent. • Two potentiators with distinct actions restore CFTR activity to rare CF mutations., The treatment of cystic fibrosis (CF) has been transformed by orally-bioavailable small molecule modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), which restore function to CF mutants. However, CFTR modulators are not available to all people with CF and better modulators are required to prevent disease progression. Here, we review selectively recent advances in CFTR folding, function and pharmacology. We highlight ensemble and single-molecule studies of CFTR folding, which provide new insight into CFTR assembly, its perturbation by CF mutations and rescue by CFTR modulators. We discuss species-dependent differences in the action of the F508del-CFTR mutation on CFTR expression, stability and function, which might influence pharmacological studies of CFTR modulators in CF animal models. Finally, we illuminate the identification of combinations of two CFTR potentiators (termed co-potentiators), which restore therapeutically-relevant levels of CFTR activity to rare CF mutations. Thus, mechanistic studies of CFTR folding, function and pharmacology inform the development of highly effective CFTR modulators.

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction., Cystic fibrosis (CF) is a lethal genetic disease that is primarily caused by misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR). Here authors show that disease-causing mutations located within the first nucleotide binding domain of CFTR have distinct effects on nascent polypeptides.

The ubiquitous ribosome-associated complex (RAC) is a chaperone that spans ribosomes, making contacts near both the polypeptide exit tunnel and the decoding center, a position prime for sensing and coordinating translation and folding. Loss of RAC is known to result in growth defects and sensitization to translational and osmotic stresses. However, the physiological substrates of RAC and the mechanism(s) by which RAC is involved in responding to specific stresses in higher eukaryotes remain obscure. The data presented here uncover an essential function of mammalian RAC in the unfolded protein response (UPR). Knockdown of RAC sensitizes mammalian cells to endoplasmic reticulum (ER) stress and selectively interferes with IRE1 branch activation. Higher-order oligomerization of the inositol-requiring enzyme 1a (IRE1α) kinase/endoribonuclease depends upon RAC. These results reveal a surveillance function for RAC in the UPR, as follows: modulating IRE1α clustering as required for endonuclease activation and splicing of the substrate Xbp1 mRNA.

Premature termination codons (PTCs) are responsible for 10–15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identify ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR)., Premature termination codon suppression therapy could be used to treat a range of genetic disorders. Here the authors present a high-throughput cell-based assay to identify anticodon engineered tRNAs with high suppression activity.

In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/β-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating β-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis.

Nontuberculous mycobacteria (NTM) cause an increasingly important chronic and debilitating lung disease in older adults. Diagnosis is often delayed, although awareness among clinicians and patients is increasing. When necessary, treatment often lasts 18-24 months and consists of three or four antibiotics that can have serious side effects. Relapses are common and commonly require resumption of prolonged therapy. Given the need for improved diagnostic techniques and clinical trials to identify new therapies or to improve existing therapies, a group of North American clinicians and researchers formed the NTM Research Consortium (NTMRC) in 2014. The NTMRC recognized the importance of including the patient voice in determining research priorities for NTM. In November 2015, patients, caregivers, patient advocates, clinical experts, and researchers gathered for a 1-day meeting in Portland, Oregon funded by the Patient-Centered Outcomes Research Institute. The meeting goal was to define patient-centered research priorities for NTM lung infections. Patients expressed frustration with the number of people who have endured years of missed diagnoses or inadequate treatment of NTM. Participants identified as top research priorities the prevention of NTM infection; approval of more effective treatments with fewer side effects and easier administration; understanding the best chest physiotherapy methods; validating and using tools to measure quality of life; and developing a disease-specific activity and severity assessment tool. Workshop participants agreed that two complementary objectives are critical to ensure the best achievable outcomes for patients: (1) additional clinician education to improve screening and diagnosis of NTM infections; and (2) development of a geographically distributed network of experts in NTM disease to offer consultation or direct therapy after a diagnosis is made.

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22 °C and increased at 28 °C, a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37 °C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 °C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.

SummaryIn eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.

Summary The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389–500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal α/β-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

Hsc70 plays a productive role during cotranslational cystic fibrosis transmembrane conductance regulator folding that is outweighed by its dominant contribution to posttranslational targeting to the ubiquitin-proteasome system. Moreover, the outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is governed by regulatory cochaperones., The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

The study identifies Hsp90 as a molecular chaperone for KATP channels. Inhibition of Hsp90 function reduces, whereas overexpression of Hsp90 enhances, channel expression at the cell surface. Hsp90 facilitates channel biogenesis by targeting the SUR1 subunit. Up-regulation of Hsp90 also enhances expression of some SUR1 mutants with folding defects., The pancreatic β-cell ATP-sensitive potassium (KATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. KATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing KATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with KATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell KATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type KATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric KATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.

A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the translocon in proximity to Sec61α. This interaction is initially weak, and TM8 spontaneously dissociates from the translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61α photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61α can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.

In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/β-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating β-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis.

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.

The past decade has witnessed remarkable advances in our understanding of aquaporin (AQP) structure and function. Much, however, remains to be learned regarding how these unique and vitally important molecules are generated in living cells. A major obstacle in this respect is that AQP biogenesis takes place in a highly specialized and relatively inaccessible environment formed by the ribosome, the Sec61 translocon and the ER membrane. This review will contrast the folding pathways of two AQP family members, AQP1 and AQP4, and attempt to explain how six TM helices can be oriented across and integrated into the ER membrane in the context of current (and somewhat conflicting) translocon models. These studies indicate that AQP biogenesis is intimately linked to translocon function and that the ribosome and translocon form a highly dynamic molecular machine that both interprets and is controlled by specific information encoded within the nascent AQP polypeptide. AQP biogenesis thus has wide ranging implications for mechanisms of translocon function and general membrane protein folding pathways.

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was15% of Stat5bWT (3.5 h versus24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

During polytopic protein biogenesis, the Sec61 translocon must rapidly orient and integrate multiple transmembrane segments (TMs) into the endoplasmic reticulum membrane. To understand this process, we examined interactions between Sec61alpha and all six TMs of the aquaporin-4 (AQP4) water channel at defined stages of synthesis using incorporated photo-cross-linking probes. Each TM interacted with and moved through the translocon in a highly ordered and sequential fashion. Strong asymmetric Sec61alpha cross-linking was observed for only one helix at a time, suggesting the presence of a single primary binding site. However, up to four TMs simultaneously contacted Sec61alpha from different molecular environments. Thus, AQP4 integration by Sec61alpha involves sequential triage of TMs from their initial portal of entry into multiple secondary sites within the translocon. This mechanism provides a means to facilitate early folding events before release into the lipid bilayer.

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.

Aquaporin-2 (AQP2) is the vasopressin-sensitive water channel that regulates water reabsorption in the distal nephron collecting duct. Inherited AQP2 mutations that disrupt folding lead to nephrogenic diabetes insipidus (NDI) by targeting newly synthesized protein for degradation in the endoplasmic reticulum (ER). During synthesis, a subset of wild-type (WT) AQP2 is covalently modified by N-linked glycosylation at residue Asn123. To investigate the affect of glycosylation, we expressed WT AQP2 and four NDI-related mutants in Xenopus laevis oocytes and compared stability of glycosylated and nonglycosylated isoforms. In all constructs, approximately 15-20% of newly synthesized AQP2 was covalently modified by N-linked glycosylation. At steady state, however, core glycosylated WT protein was nearly undetectable, whereas all mutants were found predominantly in the glycosylated form (60-70%). Pulse-chase metabolic labeling studies revealed that glycosylated isoforms of mutant AQP2 were significantly more stable than their nonglycosylated counterparts. For nonglycosylated isoforms, the half-life of WT AQP2 was significantly greater (48 h) than that of mutant AQP2 (T126M 4.1 +/- 1.0 h, A147T 4.2 +/- 0.60 h, C181W 4.5 +/- 0.50 h, R187C 6.8 +/- 1.2 h). This is consistent with rapid turnover in the ER as previously reported. In contrast, the half-lives of mutant proteins containing N-linked glycans were similar to WT (approximately 25 h), indicating that differences in steady-state glycosylation profiles are caused by increased stability of glycosylated mutant proteins. These results suggest that addition of a single N-linked oligosaccharide moiety can partially compensate for ER folding defects induced by disease-related mutations.

Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane α-helical segments in the nascent chain exit the translocon via a lateral gate that opens towards the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.

The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.

Item does not contain fulltext Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip.

Homologous aquaporin water channels utilize different folding pathways to acquire their transmembrane (TM) topology in the endoplasmic reticulum (ER). AQP4 acquires each of its six TM segments via cotranslational translocation events, whereas AQP1 is initially synthesized with four TM segments and subsequently converted into a six membrane-spanning topology. To identify sequence determinants responsible for these pathways, peptide segments from AQP1 and AQP4 were systematically exchanged. Chimeric proteins were then truncated, fused to a C-terminal translocation reporter, and topology was analyzed by protease accessibility. In each chimeric context, TM1 initiated ER targeting and translocation. However, AQP4-TM2 cotranslationally terminated translocation, while AQP1-TM2 failed to terminate translocation and passed into the ER lumen. This difference in stop transfer activity was due to two residues that altered both the length and hydrophobicity of TM2 (Asn(49) and Lys(51) in AQP1 versus Met(48) and Leu(50) in AQP4). A second peptide region was identified within the TM3-4 peptide loop that enabled AQP4-TM3 but not AQP1-TM3 to reinitiate translocation and cotranslationally span the membrane. Based on these findings, it was possible to convert AQP1 into a cotranslational biogenesis mode similar to that of AQP4 by substituting just two peptide regions at the N terminus of TM2 and the C terminus of TM3. Interestingly, each of these substitutions disrupted water channel activity. These data thus establish the structural basis for different AQP folding pathways and provide evidence that variations in cotranslational folding enable polytopic proteins to acquire and/or maintain primary sequence determinants necessary for function.

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4–6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.

Galactokinase is an essential enzyme in the metabolism of galactose. Patients with deficiencies in galactokinase exhibit early-onset cataracts. We examined the sequence of the human galactokinase gene (GK1) from 13 patients exhibiting galactokinase deficiency and identified 12 novel mutations. One of the mutations occurred in six of the 13 probands examined, and the remaining 11 were unique mutations. Expression of each of the mutant GK1 genes in Xenopus oocytes resulted in very low galactokinase activity levels. These results provide important information regarding the types of GK1 mutations that occur in the human population. Hum Mutat 15:447–453, 2000. © 2000 Wiley-Liss, Inc.

The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, misfolded and unassembled proteins in the endoplasmic reticulum (ER). Using a cell-free reticulocyte lysate system we have examined the relationship between biosynthesis and ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein with 12 predicted transmembrane segments. Our results provide direct evidence that full-length, glycosylated and membrane-integrated CFTR is a substrate for degradation and that degradation involves polyubiquitination and cytosolic proteolytic activity. CFTR ubiquitination was both temperature- and ATP-dependent. Degradation was significantly inhibited by EDTA, apyrase, and the proteasome inhibitors hemin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin beta-lactone, ALLN, and Nalpha-tosyl-L-phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin and N-tosyl lysyl chloromethyl ketone. In the presence of hemin, polyubiquitinated CFTR remained tightly associated with ER microsomes. However, membrane-bound ubiquitinated CFTR could be subsequently degraded into trichloroacetic acid-soluble fragments upon incubation in hemin-free, ATP-containing lysate. Thus ER-associated degradation of CFTR occurs via a membrane-bound, rather than cytosolic, intermediate and likely involves recruitment of degradation machinery to the ER membrane. Our data suggest a model in which the degradation of polytopic proteins such as CFTR is coupled to retrograde translocation and removal of the polypeptide from the lipid bilayer.

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.

Defects in the human GALK1 gene result in galactokinase deficiency and cataract formation. We have isolated this gene and established its structural organization. The gene contains 8 exons and spans approximately 7.3 kb of genomic DNA. The GALK1 promoter was localized and found to have many features in common with other housekeeping genes, including high GC content, several copies of the binding site for the Sp1 transcription factor, and the absence of TATA-box and CCAAT-box motifs typically present in eukaryotic Pol II promoters. Analysis by 5'-RACE PCR indicates that the GALK1 mRNA is heterogeneous at the 5' terminus, with transcription sites occurring at many locations between 21 and 61 bp upstream of the ATG start site of the coding region. In vitro translation experiments of the GALK1 cDNA indicate that the protein is cytosolic and not associated with the endoplasmic reticulum membrane.

Structure and function of kidney water channels. There is now firm evidence that water transporting proteins are expressed in renal and extrarenal tissues. In the kidney, proximal-type (CHIP28) and collecting duct (WCH-CD) water channels have been identified. We have cloned three kidney cDNAs with homology to the water channel (aquaporin) family, including a mercurial-insensitive water channel (MIWC), and a glycerol-transporting protein (GLIP) in collecting duct basolateral membrane. To elucidate water transporting mechanisms, a series of molecular and spectroscopic studies were carried out on purified CHIP28 protein and expressed chimeric and mutated CHIP28 cDNAs. The results indicate that CHIP28 transports water selectively, that CHIP28 monomers are assembled in membranes as tetramers, but that individual monomers function independently. Monomers contain multiple membrane-spanning helical domains. Based on these data and recent electron crystallography results, a model for water transport is proposed in which water moves through narrow pores located within individual CHIP28 monomers.

CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels.

Two mercurial-inhibitable water-transporting proteins have been identified: CHIP28, an erythrocyte water channel also expressed in kidney tubules and selected extrarenal epithelia, and WCH-CD, a kidney collecting duct water channel. In searching for a protein responsible for the high transcellular water permeability in lung alveolus, we cloned a 32-kDa water channel (mercurial-insensitive water channel (MIWC)) from a rat lung cDNA library with several novel features. Water permeability was strongly increased in Xenopus oocytes expressing MIWC in a mercurial-insensitive manner, in contrast to known water channels. By in situ hybridization, MIWC showed an unique distribution in cells that do not express CHIP28, including kidney papillary vasa recta, cells lining the subarachnoid space and ventricles in brain, the inner nuclear layer in retina, and the conjunctival epithelium. An alternatively spliced form of MIWC with a 165-base pair deletion in the coding sequence was also identified; relative expression of the spliced mRNA was tissue-specific. The MIWC water channel may participate in the urinary concentrating mechanism, the absorption of cerebrospinal fluid, and other physiological processes.

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.

Protein folding in cells reflects a delicate interplay between biophysical properties of the nascent polypeptide, the vectorial nature and rate of translation, molecular crowding, and cellular biosynthetic machinery. To better understand how this complex environment affects de novo folding pathways as they occur in the cell, we expressed β-barrel fluorescent proteins derived from GFP and RFP in an in vitro system that allows direct analysis of cotranslational folding intermediates. Quantitative analysis of ribosome-bound eCFP and mCherry fusion proteins revealed that productive folding exhibits a sharp threshold as the length of polypeptide from the C terminus to the ribosome peptidyltransferase center is increased. Fluorescence spectroscopy, urea denaturation, and limited protease digestion confirmed that sequestration of only 10–15 C-terminal residues within the ribosome exit tunnel effectively prevents stable barrel formation, whereas folding occurs unimpeded when the C terminus is extended beyond the ribosome exit site. Nascent FPs with 10 of the 11 β-strands outside the ribosome exit tunnel acquire a non-native conformation that is remarkably stable in diverse environments. Upon ribosome release, these structural intermediates fold efficiently with kinetics that are unaffected by the cytosolic crowding or cellular chaperones. Our results indicate that during synthesis, fluorescent protein folding is initiated cotranslationally via rapid formation of a highly stable, on-pathway structural intermediate and that the rate-limiting step of folding involves autonomous incorporation of the 11th β-strand into the mature barrel structure.

Cytomegalovirus (CMV) efficiently evades many host immune defenses and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I (MHC-I) molecules in order to evade recognition and killing of infected cells by cytotoxic CD8 + T cells. We recently showed that rhesus CMV-specific Rh178 intercepts MHC-I protein translation before interference of MHC-I maturation by homologues of the human CMV US6 family. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum, displaying a short luminal and large cytosolic domain, and that the membrane-proximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. Moreover, the transfer of amino-terminal fragments containing the MHC-I signal peptide renders recipient proteins susceptible to targeting by Rh178. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particle-dependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to SEC61-dependent protein translocation, since inhibition of MHC-I translocation by eeyarestatin 1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation.

The evolution of eukaryotes was accompanied by an increased need for intracellular communication and cellular specialization. Thus, a more complex collection of secreted and membrane proteins had to be synthesized, modified, and folded. The endoplasmic reticulum (ER) thereby became equipped with devoted enzymes and associated factors that both catalyze the production of secreted proteins and remove damaged proteins. A means to modify ER function to accommodate and destroy misfolded proteins also evolved. Not surprisingly, a growing number of human diseases are linked to various facets of ER function. Each of these topics will be discussed in this article, with an emphasis on recent reports in the literature that employed diverse models.

Cell-free expression systems provide unique tools for understanding CFTR biogenesis because they reconstitute the cellular folding environment and are readily amenable to biochemical and pharmacological manipulation. The most common system for this purpose is rabbit reticulocyte lysate (RRL), supplemented with either canine pancreatic microsomes or semi-permeabilized cells, which has yielded important insights into the folding of CFTR and its individual domains. A common problem in such studies, however, is that biogenesis of large proteins such as CFTR is often inefficient due to low translation processivity, ribosome stalling, and/or premature termination. The first part of this chapter therefore describes parameters that affect in vitro translation of CFTR in RRL. We have found that CFTR expression is uniquely dependent upon 5'- and 3'-untranslated regions (UTRs) of the mRNA. Full-length CFTR expression can be markedly increased using mRNA lacking a 5'-cap analog (G(5')ppp(5')G), whereas the reverse usually holds for smaller proteins and individual CFTR domains. In the context of the full-length mRNA, translation was further stimulated by the presence of a long 3'-UTR. The second part of this chapter describes CFTR translation in lysates derived from cultured mammalian cells including human bronchial epithelial cells. Unfortunately, mammalian cell-derived lysates showed limited ability to sustain full-length CFTR synthesis. However, they provide a unique opportunity to examine specific CFTR domains (i.e., nucleotide-binding domain 1 and transmembrane domain 1) under conditions that more closely resemble the native folding environment.

The membrane protein folding problem can be articulated by two central questions. How is protein topology established by selective peptide transport to opposite sides of the cellular membrane? And how are transmembrane segments inserted, integrated and folded within the lipid bilayer? In eukaryotes, this process usually takes place in the endoplasmic reticulum (ER) coincident with protein synthesis, and is facilitated by the translating Ribosome and the Sec61 Translocon Complex (RTC). At its core, the RTC forms a dynamic pathway through which the elongating nascent polypeptide moves as it is delivered into cytosolic, lumenal and lipid compartments. This perspective will focus on emerging evidence that the RTC functions as a protein folding machine that restricts conformational space by establishing transmembrane topology and yet provides a permissive environment that enables nascent transmembrane domains to efficiently progress down their folding energy landscape.

Cell-free expression systems provide unique tools for understanding CFTR biogenesis because they reconstitute the cellular folding environment and are readily amenable to biochemical and pharmacological manipulation. The most common system for this purpose is rabbit reticulocyte lysate (RRL), supplemented with either canine pancreatic microsomes or semi-permeabilized cells, which has yielded important insights into the folding of CFTR and its individual domains. A common problem in such studies, however, is that biogenesis of large proteins such as CFTR is often inefficient due to low translation processivity, ribosome stalling, and/or premature termination. The first part of this chapter therefore describes parameters that affect in vitro translation of CFTR in RRL. We have found that CFTR expression is uniquely dependent upon 5'- and 3'-untranslated regions (UTRs) of the mRNA. Full-length CFTR expression can be markedly increased using mRNA lacking a 5'-cap analog (G(5')ppp(5')G), whereas the reverse usually holds for smaller proteins and individual CFTR domains. In the context of the full-length mRNA, translation was further stimulated by the presence of a long 3'-UTR. The second part of this chapter describes CFTR translation in lysates derived from cultured mammalian cells including human bronchial epithelial cells. Unfortunately, mammalian cell-derived lysates showed limited ability to sustain full-length CFTR synthesis. However, they provide a unique opportunity to examine specific CFTR domains (i.e., nucleotide-binding domain 1 and transmembrane domain 1) under conditions that more closely resemble the native folding environment.

The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR's first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure-function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.

Two kidney water channels have been identified: CHIP28 in proximal tubule and thin descending limb, and WCH-CD in collecting duct apical membrane. An homologous cDNA (WCH3) was obtained from rat kidney and found to encode a 276 amino acid, 29 kDa protein with 39% amino acid identity to rat CHIP28, 50% to WCH-CD and 49% to MIP26. The WCH3 transcript of 2.5 kb was expressed exclusively in kidney and was upregulated in dehydrated rats. Cell-free translation produced an approximately 28 kDa protein. Analysis of the predicted amino acid sequence indicated a hydrophobic protein with 4-6 membrane-spanning domains, with one N-linked glycosylation site, two conserved NPA boxes common to MIP26 family proteins, and conserved residue C189 common to water channels. WCH3 is a new member of the MIP26 family of channel-forming proteins in mammalian kidney.