Department of Molecular, Cellular and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California
SURFACE EXPRESSION of ion channels and receptors on the plasma membrane is of critical importance in the control of cellular signaling and communication. Each cell contains a menagerie of plasma membrane proteins, many of them oligomeric ion channels, receptors, and cell adhesion molecules. Proper regulation of the quantity and quality of this vast collection of proteins is essential for cell health. Dysregulation has been implicated in cell malfunction and disease. Several pathways control the concentrations of proteins in the plasma membrane: protein synthesis, protein degradation, retention, and export mechanisms that govern trafficking between organelles and endocytosis/recycling. Membrane spanning proteins are synthesized in the ER where the nascent protein subunits are folded, assembled into multimeric proteins and sorted. Proteins destined for the plasma membrane are then shuttled to the Golgi, posttranslationally modified and transported to the plasma membrane for insertion. Misfolded proteins are subjected to ER-associated degradation (ERAD), in which the retained proteins are transferred into the cytosol where they are ubiquitinated and degraded by the proteasome pathway (Fig. 1) (10, 22). It is becoming evident that this ubiquitin-proteasome pathway is more than a failsafe or quality control checkpoint; it is a fundamental process involved in maintaining both protein quality and quantity (Ref. 24; see p. 1351 in this issue).
|
How do cells manage to control both protein quality and delivery? The -cell KATP channel is a massive complex with a complicated structure and a highly regulated assembly process (1, 11, 17). It is a hetero-octameric complex of two types of protein subunits: four subunits of the inward rectifier potassium channel Kir6.2 and four subunits of the sulfonylurea receptor (SUR1) (6, 19). Each Kir6.2 subunit is
45 kDa with two membrane spanning segments that bracket a pore forming region, and each SUR1 subunit is
180 kDa and spans the membrane 17 times (1, 7, 11, 17). Altogether, the KATP channel traverses the plasma membrane 76 times and has a combined molecular mass of >800 kDa. With such an intricate structure, the need for stringent quality control during biogenesis is evident.
Insights into the assembly process have revealed regulatory events at several biogenic steps in the ER, the major site of quality control for membrane proteins (8, 17, 25, 27). Kir6.2 subunits assemble as a tetramer to form the pore of the channel, and the SUR1 subunits are understood to bind as a regulatory shell surrounding the core complex (Fig. 1). An ER retention signal (an arginine-based RKR motif) is present on each subunit, which prevents trafficking of partially assembled complexes (27). Upon complete subunit coassembly, the signals are masked and the KATP channel is released to the Golgi, a process, in which 14-3-3 proteins have been implicated (17, 25, 27). In addition, SUR1 is glycosylated, a modification required for robust surface expression, implying possible quality control via the calnexin/calreticulin lectin chaperones in the ER (8, 17). Proper folding and an energetically stable structure are paramount for export.
In this issue, Yan et al. (24) report a new twist to KATP channel production: that the ubiquitin-proteasome pathway plays a key role in their biogenesis and surface expression. Their results challenge the common perception that the primary role of the proteasome is to target misfolded proteins for degradation and suggest that ERAD causes significant degradation of assembly-competent subunits, thus playing a major role in downregulation of the surface expression of membrane proteins. Yan et al. (24) demonstrate that both SUR1 and Kir6.2 subunits of the KATP channel are degraded by way of the ubiquitin-proteasome pathway, using as model systems COSm6 cells and the pancreatic -cell line INS-1. Studies by Tanaka et al. (20) suggest that KATP channels also utilize a similar pathway in cardiac cells. One of the most striking findings of Yan et al. (24) is that subunit degradation via the proteasome occurs simultaneously, and with apparently similar rates, as does receptor assembly and trafficking. Thus, as subunits are synthesized, they are concurrently degraded, with both misfolded subunits, as well as functional assembly-competent subunits becoming degraded before they have the opportunity to assemble into a stable complex that is able to exit the ER.
For a large hetero-octameric membrane protein, it is not surprising that subunit biogenesis and assembly in the endoplasmic reticulum are slow. Indeed, as recently demonstrated in a detailed kinetic study by Crane and Aguilar-Bryan (9), maturation of KATP subunits is a slow process, and subunit assembly helps to protect inward rectifier K+ 6.2 (Kir6.2) subunits from degradation. Other large membrane receptors and channels share the common characteristic of slow inefficient assembly in the ER and concurrent degradation, including cystic fibrosis transmembrane conductance regulator (13), nicotinic ACh receptor (5), connexins (3), epithelial Na+ channel (18), and voltage-gated potassium channels (16). It thus appears that proteasomal degradation may be a general mechanism that limits surface expression of oligomeric membrane proteins while ensuring that unassembled subunits are cleared from the ER during oligomeric protein construction.
A second major finding of Yan and colleagues (24) is that some disease-causing mutant subunits that are normally unable to traffic to the plasma membrane can be rescued by a combination of proteasomal inhibitors and sulfonylurea drugs. The human disease familial hyperinsulinism, also called persistent hyperinsulinemic hypoglycemia of infancy (PHHI), is caused by loss-of-function mutations of KATP channel subunits. Recent studies from Yan et al. (23) demonstrated that the PHHI mutants SUR1 A116P and V187D did not traffic normally to the plasma membrane, but their trafficking defect could be partially overcome by sulfonylurea treatment, resulting in electrophysiologically normal channels. Sulfonylurea drugs are KATP channel inhibitors used in the treatment of type II diabetes; however, in this case they presumably act as pharmacological chaperones that bind to SUR1 subunits to promote a conformation that favors subunit assembly and is recognized as being properly folded. In the present study, Yan and colleagues (24) make the discovery that plasma membrane delivery of these PHHI mutants can be increased significantly by proteasome inhibitors. Importantly, they find that channel surface expression is increased by proteasome inhibitors only in the presence of sulfonylurea drugs. These observations indicate that the critical combination of a pharmacological chaperone to aid protein folding, together with inhibition of proteasomal degradation may hold the key for successful surface expression of membrane protein subunits that have restorable folding defects.
Another important result from the study by Yan and colleagues (24) raises a caution for the potential therapeutic use of inhibitors of the proteasome pathway for proteins with severe folding defects. For one of the most common PHHI mutations, SUR1 F1388, the channels appear to have severe folding defects. Cartier et al. (4) showed previously that these channels do not properly traffic to the plasma membrane, and when studied electrophysiologically, their nucleotide sensitivity and open probabilities were aberrant. Significantly, only the milder mutations were responsive to rescue to the plasma membrane by combined treatment with proteasome inhibitors and sulfonylureas. The more severe SUR1
F1388 and SUR1 L1544P mutants were not rescued (21, 24) and instead may have accumulated as intracellular aggregates. The suggestion that proteins must be folding-competent if proteasome inhibitors are to render them amenable to rescue has important implications for potential disease therapeutics. For mutant proteins that are only mildly misfolded, then proteasome inhibitors in combination with pharmacological chaperones to aid folding may be effective at promoting trafficking; whereas for proteins with severe folding defects, the cellular state may actually be worsened if intracellular aggregates of misfolded proteins are not properly cleared.
Much remains to be learned about how protein quality control to ensure correct folding and assembly of complex oligomeric membrane proteins is balanced with effective forward trafficking of proteins to their site of action on the plasma membrane. The studies presented by Yan and colleagues (24), together with recent work on several other receptors and ion channels (3, 5, 13, 18), open up a new line of investigation into ERAD as a regulator of both quality control and quantity control. Understanding the balance between biogenesis and degradation will deepen our insight into channel and receptor trafficking diseases and could reveal strategies for restoration of protein function on the cell surface that may form the basis for new therapeutics (14, 26).
![]() |
GRANTS |
---|
![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
2. Ashcroft FM and Rorsman P. Molecular defects in insulin secretion in type-2 diabetes. Rev Endocr Metab Disord 5: 135142, 2004.[CrossRef][ISI][Medline]
3. Berthoud VM, Minogue PJ, Laing JG, and Beyer EC. Pathways for degradation of connexins and gap junctions. Cardiovasc Res 62: 256267, 2004.[CrossRef][ISI][Medline]
4. Cartier EA, Conti LR, Vandenberg CA, and Shyng SL. Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy. Proc Natl Acad Sci USA 98: 28822887, 2001.
5. Christianson JC and Green WN. Regulation of nicotinic receptor expression by the ubiquitin-proteasome system. EMBO J 23: 41564165, 2004.[CrossRef][ISI][Medline]
6. Clement JP, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, Aguilar-Bryan L, and Bryan J. Association and stoichiometry of KATP channel subunits. Neuron 18: 827838, 1997.[CrossRef][ISI][Medline]
7. Conti LR, Radeke CM, Shyng SL, and Vandenberg CA. Transmembrane topology of the sulfonylurea receptor SUR1. J Biol Chem 276: 4127041278, 2001.
8. Conti LR, Radeke CM, and Vandenberg CA. Membrane targeting of ATP-sensitive potassium channel. Effects of glycosylation on surface expression. J Biol Chem 277: 2541625422, 2002.
9. Crane A and Aguilar-Bryan L. Assembly, maturation, and turnover of KATP channel subunits. J Biol Chem 279: 90809090, 2004.
10. Ellgaard L and Helenius A. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4: 181191, 2003.[CrossRef][ISI][Medline]
11. Haider S, Antcliff JF, Proks P, Sansom MS, and Ashcroft FM. Focus on Kir6.2: a key component of the ATP-sensitive potassium channel. J Mol Cell Cardiol 38: 927936, 2005.[CrossRef][ISI][Medline]
12. Huopio H, Shyng SL, Otonkoski T, and Nichols CG. KATP channels and insulin secretion disorders. Am J Physiol Endocrinol Metab 283: E207E216, 2002.
13. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, and Riordan JR. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129135, 1995.[CrossRef][ISI][Medline]
14. McCracken AA and Brodsky JL. Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD). Bioessays 25: 868877, 2003.[CrossRef][ISI][Medline]
15. Miki T and Seino S. Roles of KATP channels as metabolic sensors in acute metabolic changes. J Mol Cell Cardiol 38: 917925, 2005.[CrossRef][ISI][Medline]
16. Myers MP, Khanna R, Lee EJ, and Papazian DM. Voltage sensor mutations differentially target misfolded K+ channel subunits to proteasomal and non-proteasomal disposal pathways. FEBS Lett 568: 110116, 2004.[CrossRef][ISI][Medline]
17. Neagoe I and Schwappach B. Pas de deux in groups of fourthe biogenesis of KATP channels. J Mol Cell Cardiol 38: 887894, 2005.[CrossRef][ISI][Medline]
18. Rotin D, Kanelis V, and Schild L. Trafficking and cell surface stability of ENaC. Am J Physiol Renal Physiol 281: F391F399, 2001.
19. Shyng S and Nichols CG. Octameric stoichiometry of the KATP channel complex. J Gen Physiol 110: 655664, 1997.
20. Tanaka H, Miake J, Notsu T, Sonyama K, Sasaki N, Iitsuka K, Kato M, Taniguchi S, Igawa O, Yoshida A, Shigemasa C, Hoshikawa Y, Kurata Y, Kuniyasu A, Nakayama H, Inagaki N, Nanba E, Shiota G, Morisaki T, Ninomiya H, Kitakaze M, and Hisatome I. Proteasomal degradation of Kir6.2 channel protein and its inhibition by a Na+ channel blocker aprindine. Biochem Biophys Res Commun 331: 10011006, 2005.[CrossRef][ISI][Medline]
21. Taschenberger G, Mougey A, Shen S, Lester LB, LaFranchi S, and Shyng SL. Identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor 1 that prevents normal trafficking and function of KATP channels. J Biol Chem 277: 1713917146, 2002.
22. Tsai B, Ye Y, and Rapoport TA. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3: 246255, 2002.[CrossRef][ISI][Medline]
23. Yan F, Lin CW, Weisiger E, Cartier EA, Taschenberger G, and Shyng SL. Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor. J Biol Chem 279: 1109611105, 2004.
24. Yan FF, Lin CW, Cartier EA, and Shyng SL. Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of -cell ATP-sensitive potassium channels. Am J Physiol Cell Physiol 289: C1351C1359, 2005.
25. Yuan H, Michelsen K, and Schwappach B. 143-3 dimers probe the assembly status of multimeric membrane proteins. Curr Biol 13: 638646, 2003.[CrossRef][ISI][Medline]
26. Zavrski I, Jakob C, Schmid P, Krebbel H, Kaiser M, Fleissner C, Rosche M, Possinger K, and Sezer O. Proteasome: an emerging target for cancer therapy. Anticancer Drugs 16: 475481, 2005.[CrossRef][ISI][Medline]
27. Zerangue N, Schwappach B, Jan YN, and Jan LY. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22: 537548, 1999.[CrossRef][ISI][Medline]
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |