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Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator

2000; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1755.2000.00921.x

1523-1755

William R. Skach,

Neonatal Respiratory Health Research

Defects in processing and trafficking of cystic fibrosis transmembrane conductance regulator. Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies. Defects in processing and trafficking of cystic fibrosis transmembrane conductance regulator. Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies. ATP binding cassette transporter superfamily adenosine 5'-triphosphate cystic fibrosis cystic fibrosis transmembrane conductance regulator ubiquitin activating enzyme ubiquitin conjugating enzyme ubiquitin ligating enzyme extracellular loop first ECL in the ER lumin epithelial sodium channel endoplasmic reticulum heat shock protein nucleotide binding domain outwardly rectifying chloride channel transmembrane first TM segment wild-type The cystic fibrosis transmembrane conductance regulator (CFTR) is a complex, polytopic membrane protein expressed in the apical membrane of selected epithelial cells. CFTR functions directly as a cAMP regulated chloride channel1Collins F.S. Cystic fibrosis: Molecular biology and therapeutic implications.Science. 1992; 256: 774-779Crossref PubMed Scopus (681) Google Scholar and also regulates the activity of other membrane proteins including the epithelial sodium channel (ENaC)2Stutts M.J. Canessa C. Olsen J. Hamrick M. Cohn J. Rossier B. Boucher R. CFTR as a cAMP-dependent regulator of sodium channels.Science. 1995; 269: 847-850Crossref PubMed Scopus (910) Google Scholar and the outwardly rectifying chloride channel (ORCC)3Schwiebert E. Egan M. Hwang T. Fulmer S. Allen S. Cutting G. Guggino W. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (578) Google Scholar. It thus plays a key role in the movement of ions and water across epithelial tissues. Not surprisingly, CFTR disruption results in a pleiotropic phenotype4Boat T. Welsh M. Beaudet A. The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1989: 2649-2680Google Scholar. The most profound effects are insufficiency of the exocrine pancreas, increase in sweat chloride concentration, male infertility and recurrent pulmonary infections. In most cystic fibrosis (CF) patients, chronic airway inflammation results in progressive pulmonary scarring, reduced lung function and ultimately, death. While CFTR is also expressed in the kidney (proximal and distal tubules, cortical collecting duct and inner medullary collecting duct5Morales M. Carroll T. Morita T. Schwiebert E. Devuyst O. Wilson P. Lopes A. Stanton B. Dietz H. Cutting G. Guggino W. Both wild type and a functional isoform of CFTR are expressed in kidney.Am J Physiol. 1996; 270 (Renal Fluid Electrolyte Physiol 39): F1038-F1048PubMed Google Scholar), only mild renal abnormalities are observed in CF patients [reviewed in6Stanton B. Cystic fibrosis transmembrane conductance regulator (CFTR) and renal function.Wein Klin Wochenschr. 1997; 109/12–13: 457-464Google Scholar. These include decreased ability to excrete a salt load, mild urinary concentrating defects, increased proximal sodium reabsorption and altered drug excretion. CFTR is also expressed in the apical membrane of renal cysts in patients with polycystic kidney disease, and may play a role in chloride and fluid secretion into the cyst lumen7Hanaoka K. Devuyst O. Schwiebert E. Wilson P. Guggino W. A role for CFTR in human autosomal dominant polycystic kidney disease.Am J Physiol. 1996; 270: C389-C399PubMed Google Scholar,8Brill S. Ross K. Davidow C. Ye M. Grantham J. Caplan M. Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.Proc Natl Acad Sci USA. 1996; 93: 10206-10211Crossref PubMed Scopus (89) Google Scholar. More than 800 mutations in the CFTR gene have been identified in CF patients9CYSTIC FIBROSIS CONSORTIUM ANALYSIS Cystic Fibrosis Mutation Data Base. 1998Google Scholar. These are broadly grouped into four classes: (I) defective protein synthesis; (II) defective protein processing; (III) defective ion conduction; and (IV) defective regulation of channel gating10Welsh M. Smith A. Molecular mechanisn of CFTR chloride channel dysfunction in cystic fibrosis.Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1152) Google Scholar. Defective processing is by far the most common mechanism of protein disruption, accounting for more than 2/3 of clinical CF cases11Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'riordan C.R. Smith A.E. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1359) Google Scholar. While these latter patients synthesize adequate amounts of functional CFTR protein, the mutant protein is rapidly degraded prior to reaching the plasma membrane. To understand the molecular basis of CF it will therefore be necessary to define the molecular mechanism(s) by which CFTR is folded, assembled and packaged into cellular membranes and trafficked through cells. CFTR is a member of the ATP binding cassette (ABC) transporter superfamily12Riordan J.R. Rommens J.M. Kerem B-S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Collins F.S. Tsui L-C. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1072Crossref PubMed Scopus (5604) Google Scholar. It contains two hydrophobic domains (each with six predicted transmembrane (TM) segments), two cytosolic nucleotide binding domains (NBDs) and a cytosolic regulatory (R) domain. Like most eukaryotic membrane proteins, CFTR is synthesized and assembled in the endoplasmic reticulum (ER). During the earliest steps in this process, nascent chain-ribosome complexes are targeted to the ER membrane, and TM segments are precisely oriented and integrated into the lipid bilayer13Lu Y. Xiong X. Bragin A. Kamani K. Skach W. Co- and posttranslational mechanisms direct CFTR N-terminus transmembrane assembly.J Biol Chem. 1997; 273: 568-576Crossref Scopus (87) Google Scholar. Additional biogenesis events involve the packing of transmembrane helices, folding of cytosolic domains, and finally, assembly of these domains into a mature tertiary structure14Ostedgaard L. Rich D. Deberg L. Welsh M. Association of domains within the cystic fibrosis transmembrane conductance regulator.Biochemistry. 1997; 36: 1287-1294Crossref PubMed Scopus (59) Google Scholar. This process is mediated by specialized cellular machinery that includes the Sec61 translocation complex and cytosolic (hsp70, hsp40) as well as ER (calnexin) chaperones that assist folding and prevent aggregation of folding intermediates15Pind S. Riordan J. Williams D. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator.J Biol Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar,16Yang Y. Janach S. Cohn J. Wilson J. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment.Proc Natl Acad Sci USA. 1993; 90: 9480-9484Crossref PubMed Scopus (272) Google Scholar. CFTR maturation is thus a stepwise and compartmentalized process that coordinates folding of different protein domains in the lipid environment of the ER membrane, the oxidizing environment of the ER lumen and the reducing environment of the cytosol Figure 1. At the center of this process is a stringent quality control mechanism capable of discriminating normally folded from abnormally folded proteins17Kopito R. ER quality control: The cytoplasmic connection.Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar. Quality control machinery thus prevents misfolded CFTR from exiting the ER compartment and is responsible for its degradation via the cytosolic ubiquitin/proteasome pathway18Ward C. Omura C. Kopito R. Degradation of CFTR by the ubiquitin-proteosome pathway.Cell. 1995; 83: 121-128Abstract Full Text PDF PubMed Scopus (1088) Google Scholar,19Jensen T. Loo M. Pind S. Williams D. Goldberg A. Riordan J. Multiple proteolytic systems, including the proteosome, contribute to CFTR processing.Cell. 1995; 83: 129-136Abstract Full Text PDF PubMed Scopus (742) Google Scholar. This article reviews recent studies that address three questions related to the underlying defect in CFTR processing and trafficking. How is WT CFTR assembled into the ER membrane, and how do inherited mutations affect this process? What general structural features in mutant CFTR might serve to alert cellular quality control machinery? Finally, how is misfolded CFTR delivered to cytosolic proteases? Conventional models predict that the transmembrane topology of polytopic proteins is established through the action of sequential signal and stop transfer sequences as the nascent chain emerges from the ribosome24Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J. Molecular Biology of the Cell. New York, Garland Publishing, 1994: 577-589Google Scholar. Signal sequences function to target nascent chains to the ER, facilitate ribosome binding to the membrane, and open a large aqueous translocation channel through which the elongating nascent chain moves to reach the ER lumen25Walter P. Blobel G. Translocation of proteins across the endoplasmic reticulum II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro assembled polysomes synthesizing secretory protein.J Cell Biol. 1981; 91: 551-556Crossref PubMed Scopus (244) Google Scholar, 26Crowley K. Liao S. Worrell V. Reinhart G. Johnson A. Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore.Cell. 1994; 78: 461-471Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 27Simon S.M. Blobel G. A protein-conducting channel in the endoplasmic reticulum.Cell. 1991; 65: 371-380Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 28Jungnickel B. Rapoport T. A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane.Cell. 1995; 82: 261-270Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 29Mothes W. Heinrich S. Graf R. Nilsson I. Von Heijne G. Brunner J. Rapoport T. Molecular mechanism of membrane protein integration into the endoplasmic reticulum.Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar. Stop transfer sequences terminate ongoing translocation, disrupt the ribosome-membrane junction, close the translocon, and direct the hydrophobic TM helix laterally out of the translocon and into the lipid bilayer29Mothes W. Heinrich S. Graf R. Nilsson I. Von Heijne G. Brunner J. Rapoport T. Molecular mechanism of membrane protein integration into the endoplasmic reticulum.Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 30Liao S. Lin J. Do H. Johnson A. Both lumenal and cytosolic gating of the aqueous transocon pore are regulated from inside the ribosome during membrane protein integration.Cell. 1997; 90: 31-42Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 31Do H. Falcone D. Lin J. Andrews D. Johnson A. The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process.Cell. 1996; 85: 369-378Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar. A large number of molecular events must therefore be precisely coordinated as polytopic proteins such as CFTR are "stitched" into the ER membrane. If CFTR followed a conventional biogenesis model, then the first TM segment (TM1) should encode signal sequence activity capable of orienting the N-terminus in the cytosol and the first extracellular loop (ECL1) in the ER lumen. Surprisingly, when TM1 signal sequence activity was tested in a defined heterologous cassette, TM1 was unable to efficiently initiate translocation or span the membrane13Lu Y. Xiong X. Bragin A. Kamani K. Skach W. Co- and posttranslational mechanisms direct CFTR N-terminus transmembrane assembly.J Biol Chem. 1997; 273: 568-576Crossref Scopus (87) Google Scholar. This suggested either that CFTR assembly into the ER membrane was inefficient, or that topogenic information in addition to TM1 was required for N-terminus transmembrane assembly. Subsequent analysis confirmed the latter prediction by demonstrating that TM2 also functioned as a signal sequence with translocation specificity complimentary to that of TM1. Moreover, by simultaneously disrupting signal sequence activities of TM1 and TM2, it was shown that TM2 was able to independently orient TM1 and ECL1 in the ER membrane after this region had been synthesized in the cytosol. This post-translational translocation activity of TM2 was ribosome dependent, indicating that TM2, like TM1, utilized established translocation machinery (for example, signal recognition particle) for ER targeting32Ng D. Brown J. Walter P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane.J Cell Biol. 1996; 134: 269-278Crossref PubMed Scopus (354) Google Scholar. Mutagenesis studies of TM1 and TM2 thus define two alternate translocation pathways by which CFTR acquires its proper N-terminus transmembrane topology (diagrammed in Figure 2). For a minority of nascent chains, TM1 functions as a signal sequence to initiate translocation into the ER lumen. In these chains, TM2 stop transfer activity terminates ongoing translocation and establishes the membrane boundaries of TM1, TM2 and ECL1. In chains where TM1 fails to start translocation, however, TM1 and TM2 emerge from the ribosome into the cytosol where TM2 initiates translocation of its N-terminus flanking residues. Here, TM1 functions in the capacity of a stop transfer sequence and is positioned post-translationally into its proper orientation. In this manner, TM2 provides a backup mechanism for ensuring proper topology in chains where TM1-mediated translocation has failed. Further analysis indicated that two charged residues located within the hydrophobic membrane spanning core of TM1 (E92 and K95) were responsible for the weak TM1 signal sequence activity. Mutating these residues to alanine markedly improved the ability of TM1 to direct translocation13Lu Y. Xiong X. Bragin A. Kamani K. Skach W. Co- and posttranslational mechanisms direct CFTR N-terminus transmembrane assembly.J Biol Chem. 1997; 273: 568-576Crossref Scopus (87) Google Scholar but completely disrupted CFTR chloride channel activity in Xenopus oocytes (unpublished observations). Thus, for CFTR, structural features required for protein function (such as residues E92 and K95) directly conflict with structural features necessary to direct CFTR topology via the cotranslational pathway Figure 2. The presence of TM2 signal sequence activity, by providing an alternate mechanism to ensure CFTR topology, therefore enables TM1 to contain the necessary charged residues. This increased sequence diversity within TM1 would not have been possible if CFTR biogenesis were restricted solely to the conventional mode of biogenesis. While CFTR provides the first example of this type of redundancy in topogenic pathways, it seems likely that other polytopic proteins, particularly those with specialized structural requirements, will exhibit additional variations in transmembrane assembly. Two CF mutations, G85E and G91R, each introduce an additional charged residue within the hydrophobic core of TM1. These mutations also disrupted CFTR chloride efflux in microinjected Xenopus oocytes by preventing newly synthesized protein from exiting the ER33Xiong X. Bragin A. Widdicombe J. Cohn J. Skach W. Structural cues involved in ER degradation of G85E and G91R mutant CFTR.J Clin Invest. 1997; 100: 1079-1088Crossref PubMed Scopus (55) Google Scholar. This suggested that G85E and G91R CFTR mutants failed to fold properly and were recognized by ER quality control machinery similar to the common αδF508 mutant11Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'riordan C.R. Smith A.E. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1359) Google Scholar. To understand how charged residues within TM1 influenced CFTR folding, we compared N-terminus transmembrane assembly and topology in WT and mutant chains. Topologic analysis revealed that each mutation completely eliminated TM1 signal sequence activity but had no effect on CFTR topology13Lu Y. Xiong X. Bragin A. Kamani K. Skach W. Co- and posttranslational mechanisms direct CFTR N-terminus transmembrane assembly.J Biol Chem. 1997; 273: 568-576Crossref Scopus (87) Google Scholar,33Xiong X. Bragin A. Widdicombe J. Cohn J. Skach W. Structural cues involved in ER degradation of G85E and G91R mutant CFTR.J Clin Invest. 1997; 100: 1079-1088Crossref PubMed Scopus (55) Google Scholar. Thus, in these mutant chains, TM2 was entirely responsible for directing translocation of ECL1. More importantly, because mutant TM1 and TM2 each spanned the membrane in their native orientations, ER quality control machinery must have been able to detect the presence of the aberrant charged residues localized within the plane of the lipid bilayer. To determine how this might occur, WT and mutant CFTR constructs were truncated after the second transmembrane segment, the first transmembrane domain (TM6), NBD1 or the R domain. Expression of these constructs expressed in Xenopus oocytes demonstrated that cellular quality control machinery was effectively able to distinguish WT from mutant chains only after synthesis of the R domain had been completed34Xiong X. Bragin A. Skach W. Evidence that degradation of mutant CFTR involves interactions between multiple domains.Pediatric Pulmonol. 1996; Suppl 13: 210Google Scholar. These studies demonstrated that in order for CFTR to acquire a stable structure in the ER membrane, multiple protein domains must be synthesized and properly assembled. In addition, they indicated that G85E and G91R mutations likely interfered with late, rather than early, assembly events required for CFTR tertiary structure. A subtle alteration in the first transmembrane domain such as the insertion of a charged residue may thus indirectly influence folding interactions at distant sites in the molecule. This provides an intriguing model as to how mutant proteins might be recognized by ER quality control machinery, and how mutations in diverse regions of CFTR could give rise to similar trafficking phenotypes. If ER quality control machinery recognized structural interfaces between CFTR subdomains, then subtle structural changes that influence the strength and/or kinetics of these interactions could be recognized by quality control machinery in much the same manner as unassembled oligomeric subunits. ER quality control machinery would therefore not be required to recognize each local structural perturbation, but rather it might serve to monitor more global aspects of protein compaction. Finally, it should be noted that "abnormal" protein folding in terms of ER quality control is operational and entirely based on a cellular response, namely ER associated degradation17Kopito R. ER quality control: The cytoplasmic connection.Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar. While it is often tempting to view the acquisition of protein function as a criteria for "normal" folding, in the case of CFTR this is not necessarily correct. CFTR protein containing the αδF508 mutation is clearly capable of forming cAMP gated chloride channels with nearly normal conduction properties35Pasyk E. Foskett K. Mutant (αδF508) cystic fibrosis transmembrane conductance regulator Cl– channel is functional when retained in the endoplasmic reticulum of mammalian cells.J Biol Chem. 1995; 270: 12347-12350Crossref PubMed Scopus (125) Google Scholar. Yet essentially 100% of αδF508 CFTR is degraded in the ER. Conversely, WT CFTR protein truncated after the R domain at residue #836 is nearly as stable as full length protein in Xenopus oocytes33Xiong X. Bragin A. Widdicombe J. Cohn J. Skach W. Structural cues involved in ER degradation of G85E and G91R mutant CFTR.J Clin Invest. 1997; 100: 1079-1088Crossref PubMed Scopus (55) Google Scholar, yet its chloride channel activity is <5% of wild-type (unpublished observations). Thus, protein maturation in a functional sense may be distinct from structural maturation as determined by ER quality control machinery. This process is further complicated by observations that the efficiency of intracellular trafficking differs markedly between cell systems. In mammalian cells, 80% of WT and ∼99% of αδ F508 CFTR is degraded in the ER, while in Xenopus oocytes, <10% of WT and ∼80% of αδF508 is degraded in the ER34Xiong X. Bragin A. Skach W. Evidence that degradation of mutant CFTR involves interactions between multiple domains.Pediatric Pulmonol. 1996; Suppl 13: 210Google Scholar. It is unknown whether these differences in intracellular trafficking reflect different folding efficiencies or alternatively, different stringencies in quality control systems. In either case, understanding the relationship between CFTR quality control and CFTR functional maturation will likely require detailed structural studies and the identification of cellular components responsible for discriminating subtle structural differences. The hallmark of abnormal CFTR processing and trafficking is rapid degradation of CFTR protein in a pre-Golgi and lysosome-independent compartment. Surprisingly, several studies have now demonstrated that the cytosolic ubiquitin-proteasome pathway plays a key role in ER associated degradation not only of CFTR, but also of a wide variety of misfolded secretory, bitopic and polytopic protein substrates17Kopito R. ER quality control: The cytoplasmic connection.Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar,36Sommer T. Wolf D. Endoplasmic reticulum: Reverse protein flow of no return.FASEB J. 1997; 11: 1227-1233Crossref PubMed Scopus (216) Google Scholar. In the ubiquitin proteasome pathway, substrates are first modified by covalent addition of multiple ubiquitin moieties through the action of cytosolic (and/or membrane bound) ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes [reviewed in37Ciechanover A. The ubiquitin-proteasome pathway.Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1546) Google Scholar. Polyubiquitinated proteins are then recognized by the cytosolic 26S proteasome complex; ubiquitin chains are removed; and the substrate is digested into small peptide fragments. These observations require that cytosolic degradation machinery gains access to proteins in the lumen (or membrane) of the ER, and suggest that translocation across the ER membrane is a bidirectional process that is regulated in part by the folded state of a given protein33Xiong X. Bragin A. Widdicombe J. Cohn J. Skach W. Structural cues involved in ER degradation of G85E and G91R mutant CFTR.J Clin Invest. 1997; 100: 1079-1088Crossref PubMed Scopus (55) Google Scholar,38Brodsky J. McCracken A. ER-asociated and proteasome-mediated protein degradation: How two topologically restricted events came together.Trends Cell Biol. 1997; 7: 151-155Abstract Full Text PDF PubMed Scopus (183) Google Scholar. The degradation of polytopic proteins by cytosolic proteases poses an additional topologic challenge in that multiple transmembrane helices must be removed from the bilayer. Then, at what stage of biogenesis is CFTR recognized for degradation, and once recognized, how is CFTR delivered to the cytosolic proteolytic complex? Recently, Sato, Ward and Kopito used an in vitro expression system to demonstrate that CFTR ubiquitination might actually begin prior to the completion of protein synthesis39Sato S. Ward C. Kopito R. Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator.In Vitro J Biol Chem. 1998; 273: 7189-7192Crossref PubMed Scopus (115) Google Scholar. Using a similar rabbit reticulocyte lysate-based expression system, we showed that full length and membrane integrated CFTR is also a substrate for polyubiquitination Figure 340Xiong X. Chong E. Skach W. Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane.J Biol Chem. 1999; 274: 2616-2624Crossref PubMed Scopus (95) Google Scholar. CFTR ubiquitination required cytosolic components as well as ATP. By allowing ubiquitination to occur in the presence of the proteasome inhibitor hemin, we demonstrated that ubiquitinated CFTR remained tightly associated with the ER membrane until it was degraded into trichloroacetic acid-soluble fragments. Furthermore, pre-ubiquitinated, membrane-bound CFTR could be degraded only in the presence of additional cytosol40Xiong X. Chong E. Skach W. Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane.J Biol Chem. 1999; 274: 2616-2624Crossref PubMed Scopus (95) Google Scholar. These results suggest that polyubiquitinated CFTR is involved in recruiting cytosolic degradation machinery directly to the ER membrane, consistent with studies by Rivett, Palmer and Knecht, which demonstrate that proteasomes are bound to the ER in living cells41Rivett A.J. Palmer A. Knecht E. Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.J Histochem Cytochem. 1992; 40: 1165-1172Crossref PubMed Scopus (126) Google Scholar. Because hemin inhibits the proteasome by blocking ATPase activities within the 19 S subunit (PA700)42Hoffman L. Rechsteiner M. Nucleotidase activities of the 26 S proteasome and its regulatory complex.J Biol Chem. 1996; 271: 32538-32545Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, it is also possible that the unfolding activity of the proteasome itself might be involved in extracting CFTR from the membrane. This would explain our observation that CFTR degradation was tightly coupled to extraction of TM helices from the lipid bilayer. It would also explain why proteasome inhibitors such as peptide aldehydes and/or lactacystin that directly inactivate the catalytic active site in the proteasome, but do not effect ATPase activity42Hoffman L. Rechsteiner M. Nucleotidase activities of the 26 S proteasome and its regulatory complex.J Biol Chem. 1996; 271: 32538-32545Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, might give rise to cytosolic intermediates of ER degradation substrates. In the past decade medical research has uncovered the genetic basis for an expanding group of ion channelopathies. A significant challenge in the next decade will be to decipher the underlying cellular and metabolic pathways that are influenced by these mutations. This will involve: (1) identifying the molecular components and steps that regulate normal biosynthetic processes; (2) defining how specific mutations influence these pathways; and (3) devising strategies for controlled manipulation of these pathways in human disease. In this regard, CF has served as a key example for understanding fundamental mechanisms of protein biogenesis, folding and degradation. It seems highly likely that these studies will have far reaching implications, and that future efforts to understand and correct the CF defect at the cellular level will impact an ever growing variety of ion channelopathies.