The Role of Monoubiquitination in Endocytic Degradation of Human Ether-a-go-go-related Gene (hERG) Channels under Low K+ Conditions

Abstract

A reduction in extracellular K+ concentration ([K+]o) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (IKr) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K+ conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K+-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K+ conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K+ conditions. A reduction in extracellular K+ concentration ([K+]o) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (IKr) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K+ conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K+-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K+ conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K+ conditions. IntroductionThe human ether-a-go-go-related gene (hERG) 2The abbreviations used are: hERG, the human ether-a-go-go-related gene; IKr, the rapidly activating delayed rectifier potassium channel; LQTS, long QT syndrome; Ub, ubiquitin; UbKO, lysine-less Ub mutant; MVB, multivesicular body; hVps24, Homo sapiens vacuolar protein sorting 24 homolog (Saccharomyces cerevisiae); STAMBP, signal transducing adaptor molecule-binding protein; MEM, minimum essential medium; IP, immunoprecipitation; ESCRT, endosomal sorting complex required for transport. encodes the pore-forming subunits of the rapidly activating delayed rectifier K+ channels (IKr) (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2132) Google Scholar, 2Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1082) Google Scholar). IKr is important for cardiac repolarization, and its reduction causes long QT syndrome (LQTS), a disorder that predisposes individuals to life-threatening arrhythmias (3Sanguinetti M.C. Tristani-Firouzi M. Nature. 2006; 440: 463-469Crossref PubMed Scopus (1160) Google Scholar). Mutations in hERG cause type 2 inherited long QT syndrome (LQT2), and a diverse variety of medications can block hERG and cause acquired LQTS as a cardiac side effect (4Keating M.T. Sanguinetti M.C. Cell. 2001; 104: 569-580Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). In addition, a reduction in extracellular K+ concentration ([K+]o) exacerbates LQTS (5Roden D.M. Woosley R.L. Primm R.K. Am. Heart J. 1986; 111: 1088-1093Crossref PubMed Scopus (424) Google Scholar). Using an in vivo rabbit model, we previously demonstrated that hypokalemia chronically reduces IKr and prolongs the action potential duration and the QT intervals of electrocardiograms. We further showed that extracellular K+ (K+o) is required for hERG function and membrane stability; upon depletion of K+o, hERG channels enter into a non-conducting state within minutes and are subsequently internalized and degraded within hours (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar, 7Massaeli H. Guo J. Xu J. Zhang S. Circ. Res. 2010; 106: 1072-1082Crossref PubMed Scopus (36) Google Scholar). We have also demonstrated an involvement of caveolin in low K+-induced internalization of cell surface hERG channels (8Massaeli H. Sun T. Li X. Shallow H. Wu J. Xu J. Li W. Hanson C. Guo J. Zhang S. J. Biol. Chem. 2010; 285: 27259-27264Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). However, the cellular machinery for hERG endocytic degradation is largely unknown. In particular, although ubiquitin (Ub) is found to be involved in the internalization of cell surface hERG channels (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar, 7Massaeli H. Guo J. Xu J. Zhang S. Circ. Res. 2010; 106: 1072-1082Crossref PubMed Scopus (36) Google Scholar), the nature of Ub-hERG interactions is not known.Ub is a protein with a highly conserved sequence of 76 amino acids found in every eukaryotic cell. The covalent binding of Ub to target proteins is known as ubiquitination, a process well known for labeling proteins for degradation (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 10Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar). Appendage of a single Ub moiety leads to monoubiquitination, whereas attachment of a chain of Ub leads to polyubiquitination. Although polyubiquitination targets proteins for proteasomal degradation (11Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), monoubiquitination targets membrane proteins for internalization and lysosomal degradation. As an internalization signal of membrane proteins, monoubiquitination is well described in yeast (12Galan J.M. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (320) Google Scholar, 13Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 14Lucero P. Peñalver E. Vela L. Lagunas R. J. Bacteriol. 2000; 182: 241-243Crossref PubMed Scopus (32) Google Scholar). In mammals, evidence of this connection is also accumulating (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 15Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (652) Google Scholar, 16Patrick G.N. Bingol B. Weld H.A. Schuman E.M. Curr. Biol. 2003; 13: 2073-2081Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 17Gupta-Rossi N. Six E. LeBail O. Logeat F. Chastagner P. Olry A. Israël A. Brou C. J. Cell Biol. 2004; 166: 73-83Crossref PubMed Scopus (181) Google Scholar, 18d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (201) Google Scholar, 19Sigismund S. Woelk T. Puri C. Maspero E. Tacchetti C. Transidico P. Di Fiore P.P. Polo S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2760-2765Crossref PubMed Scopus (653) Google Scholar, 20Chen H. De Camilli P. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2766-2771Crossref PubMed Scopus (122) Google Scholar, 21Leithe E. Kjenseth A. Sirnes S. Stenmark H. Brech A. Rivedal E. J. Cell Sci. 2009; 122: 3883-3893Crossref PubMed Scopus (73) Google Scholar, 22Lin D.H. Yue P. Pan C.Y. Sun P. Zhang X. Han Z. Roos M. Caplan M. Giebisch G. Wang W.H. J. Biol. Chem. 2009; 284: 29614-29624Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).In the present study, using biochemical and electrophysiological approaches, we demonstrated, for the first time, that under low K+ conditions monoubiquitination of cell surface hERG channels directs the channel internalization and degradation through the multivesicular body (MVB) pathway, and Ub is released from the internalized channel prior to the channel entry into lysosomes for further degradation.DISCUSSIONIKr is important for cardiac action potential repolarization, and its reduction causes LQTS (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2132) Google Scholar, 2Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1082) Google Scholar, 4Keating M.T. Sanguinetti M.C. Cell. 2001; 104: 569-580Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 36Sanguinetti M.C. Jurkiewicz N.K. J. Gen. Physiol. 1990; 96: 195-215Crossref PubMed Scopus (1340) Google Scholar). A reduction in serum K+ concentration (hypokalemia) precipitates LQTS (5Roden D.M. Woosley R.L. Primm R.K. Am. Heart J. 1986; 111: 1088-1093Crossref PubMed Scopus (424) Google Scholar). We recently showed that hypokalemia enhances cell surface hERG internalization and degradation and thus identified a novel mechanism through which hypokalemia induces cardiac arrhythmias (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar). Although our work showed that Ub is involved in endocytic degradation of cell surface hERG channels under low K+ conditions (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar, 7Massaeli H. Guo J. Xu J. Zhang S. Circ. Res. 2010; 106: 1072-1082Crossref PubMed Scopus (36) Google Scholar), the nature of Ub-hERG interactions is unknown. The present study demonstrated that monoubiquitination mediates degradation of the voltage-gated K+ channel hERG under low K+ conditions. This conclusion is supported by the observations that overexpression of UbKO enhanced the degradation of the 155-kDa hERG channels (FIGURE 2, FIGURE 3) and that UbKO preferentially associated with hERG channels under low K+ conditions in the presence of lactacystin (Fig. 6). Ub can polymerize by binding to other Ub molecules at lysine residues (37Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1009) Google Scholar); UbKO has all lysine residues mutated to arginine residues and can only support monoubiquitination. Hence, UbKO is a useful tool in evaluating monoubiquitin modification of cargo proteins (24Lim K.L. Chew K.C. Tan J.M. Wang C. Chung K.K. Zhang Y. Tanaka Y. Smith W. Engelender S. Ross C.A. Dawson V.L. Dawson T.M. J. Neurosci. 2005; 25: 2002-2009Crossref PubMed Scopus (439) Google Scholar, 25Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).Numerous cellular proteins are post-translationally modified by the addition of the small modifier protein Ub for maintaining cellular health. Whereas polyubiquitination usually leads to proteasomal degradation of various cellular proteins, monoubiquitination regulates endocytic degradation of membrane receptors in lysosomes (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 10Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar). The monoubiquitination-controlled attenuation of the receptor-mediated signaling pathways plays a central role in maintaining signaling homeostasis. For example, epidermal growth factor receptor, the epithelial sodium channel, the inward rectifier potassium channel ROMK1, and receptor tyrosine kinases are all monoubiquitinated, and monoubiquitin acts as a signal for internalization of targeted proteins through sorting in MVBs for lysosomal degradation (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 28Mosesson Y. Shtiegman K. Katz M. Zwang Y. Vereb G. Szollosi J. Yarden Y. J. Biol. Chem. 2003; 278: 21323-21326Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). MVBs are formed when segments of the endosomal membrane endocytose and become vesicles within a larger endosomal body. ESCRT-I first binds both Ub and phosphatidylinositol 3-phosphate and initiates the sorting mechanism (37Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1009) Google Scholar). ESCRT-II then brings ESCRT-III to the desired site on the endosomal membrane where they sequester the MVB cargo (37Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1009) Google Scholar). The morphological changes in the intraluminal vesicles reflect the sorting of ubiquitinated cargo toward them (31Wollert T. Wunder C. Lippincott-Schwartz J. Hurley J.H. Nature. 2009; 458: 172-177Crossref PubMed Scopus (458) Google Scholar). Vps24 is an important member of ESCRT-III required for cargo protein sorting. As shown in Fig. 5, hVps24 is involved in the degradation of mature hERG channels in low K+ conditions. Overexpression of hVps24 enhanced the 155-kDa hERG degradation in low K+, whereas knockdown of hVps24 impeded this process. The hVps24 protein displays predominantly plasma membrane and vesicular localization consistent with its role in the formation of MVBs (32Muzioł T. Pineda-Molina E. Ravelli R.B. Zamborlini A. Usami Y. Göttlinger H. Weissenhorn W. Dev. Cell. 2006; 10: 821-830Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Our data also showed that internalized hERG channels colocalized with the hVps24 protein, a marker protein of MVBs (Fig. 4). Because hVps24 is involved in the MVB sorting of internalized cargo proteins, depletion of hVps24 obstructs hERG sorting in MVBs, leading to an accumulation of internalized hERG channels in endosomes. Such accumulation may impede hERG endocytosis through either an increased endosome recycling to the plasma membrane or an unknown feedback mechanism.Because UbKO is a chain elongation-defective Ub mutant, it causes premature termination of polyubiquitin chains (Fig. 2C) and only supports monoubiquitination of target proteins (24Lim K.L. Chew K.C. Tan J.M. Wang C. Chung K.K. Zhang Y. Tanaka Y. Smith W. Engelender S. Ross C.A. Dawson V.L. Dawson T.M. J. Neurosci. 2005; 25: 2002-2009Crossref PubMed Scopus (439) Google Scholar, 25Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Our data showed that UbKO expression significantly accelerated low K+-induced endocytic degradation of the mature hERG channels (FIGURE 2, FIGURE 3). Thus, polyubiquitination may not play a decisive role in low K+-induced hERG internalization. On the other hand, low K+-induced hERG internalization can be impeded by the proteasomal inhibitor lactacystin (FIGURE 6, FIGURE 7) as well as MG132 (data not shown). The exact mechanisms for proteasomal activity in the MVB/lysosomal degradation of hERG channels under low K+ conditions warrant further investigation and may be related to the fact that proteasomal activity is required for the sorting of ubiquitinated cargo proteins in MVBs (38Hurley J.H. Curr. Opin. Cell Biol. 2008; 20: 4-11Crossref PubMed Scopus (336) Google Scholar). Interestingly, our data demonstrated that lactacystin not only prevented 155-kDa hERG degradation but also shifted the 155-kDa hERG toward a (Fig. suggesting that mature hERG channels are monoubiquitinated in the presence of our data that hERG channels are monoubiquitinated and We that monoubiquitination triggers internalization of hERG channels, and deubiquitination is subsequently required for hERG sorting and degradation in (Fig. The fact that lactacystin degradation of mature hERG channels that are ubiquitinated a as a proteasomal lactacystin hERG degradation in the MVB/lysosomal by deubiquitination of hERG channels. proteasomal activity has also shown to be required for degradation of a of membrane proteins that are internalized the pathway. membrane proteins receptors G.N. Bingol B. Weld H.A. Schuman E.M. Curr. Biol. 2003; 13: 2073-2081Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), A receptor T. Traffic. 2008; 9: PubMed Scopus Google Scholar), growth receptor P. R. C.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), channel M. L. Wu Y. A. Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar), and channel E.M. Cell Res. 1997; PubMed Scopus Google Scholar). be to the role of deubiquitination in the degradation of membrane data using (Fig. and (Fig. 7) further demonstrated that internalized hERG not ubiquitinated when it lysosomes in the presence of lysosomal inhibitor bafilomycin data that deubiquitination of hERG sorting and degradation. Our data on the deubiquitination enzyme support this (Fig. and that the monoubiquitinated and internalized 155-kDa hERG is subsequently and MVB sorting. Thus, Ub molecules are released from mature hERG channels prior to to lysosomes for degradation. Because deubiquitination of mature hERG is to at a of endocytosis cargo sorting in MVBs where is by the ubiquitinated not the of monoubiquitination of cell surface hERG channels by low K+ plays a role in endocytosis of receptor 2 B. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar). In our data showed that monoubiquitination at the of the directs the mature hERG channel for MVB and the internalized mature hERG channel is in a state when it of the essential for cell We previously showed that extracellular K+ is a for hERG function and membrane stability; under low K+ conditions, cell surface hERG channels enter into a non-conducting a of the This may cause the channel to be by Ub, which triggers the channel internalization through a endocytic (8Massaeli H. Sun T. Li X. Shallow H. Wu J. Xu J. Li W. Hanson C. Guo J. Zhang S. J. Biol. Chem. 2010; 285: 27259-27264Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Our present study that attachment of a single Ub moiety is to internalization of cell surface hERG the internalized mature hERG channels are by the in MVBs and deubiquitination prior to entry into lysosomes for we have demonstrated that the cell surface voltage-gated potassium channel hERG is internalized and degraded through a sorting in a under low K+ conditions. This our of channel and endocytic degradation of plasma membrane IntroductionThe human ether-a-go-go-related gene (hERG) 2The abbreviations used are: hERG, the human ether-a-go-go-related gene; IKr, the rapidly activating delayed rectifier potassium channel; LQTS, long QT syndrome; Ub, ubiquitin; UbKO, lysine-less Ub mutant; MVB, multivesicular body; hVps24, Homo sapiens vacuolar protein sorting 24 homolog (Saccharomyces cerevisiae); STAMBP, signal transducing adaptor molecule-binding protein; MEM, minimum essential medium; IP, immunoprecipitation; ESCRT, endosomal sorting complex required for transport. encodes the pore-forming subunits of the rapidly activating delayed rectifier K+ channels (IKr) (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2132) Google Scholar, 2Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1082) Google Scholar). IKr is important for cardiac repolarization, and its reduction causes long QT syndrome (LQTS), a disorder that predisposes individuals to life-threatening arrhythmias (3Sanguinetti M.C. Tristani-Firouzi M. Nature. 2006; 440: 463-469Crossref PubMed Scopus (1160) Google Scholar). Mutations in hERG cause type 2 inherited long QT syndrome (LQT2), and a diverse variety of medications can block hERG and cause acquired LQTS as a cardiac side effect (4Keating M.T. Sanguinetti M.C. Cell. 2001; 104: 569-580Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). In addition, a reduction in extracellular K+ concentration ([K+]o) exacerbates LQTS (5Roden D.M. Woosley R.L. Primm R.K. Am. Heart J. 1986; 111: 1088-1093Crossref PubMed Scopus (424) Google Scholar). Using an in vivo rabbit model, we previously demonstrated that hypokalemia chronically reduces IKr and prolongs the action potential duration and the QT intervals of electrocardiograms. We further showed that extracellular K+ (K+o) is required for hERG function and membrane stability; upon depletion of K+o, hERG channels enter into a non-conducting state within minutes and are subsequently internalized and degraded within hours (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar, 7Massaeli H. Guo J. Xu J. Zhang S. Circ. Res. 2010; 106: 1072-1082Crossref PubMed Scopus (36) Google Scholar). We have also demonstrated an involvement of caveolin in low K+-induced internalization of cell surface hERG channels (8Massaeli H. Sun T. Li X. Shallow H. Wu J. Xu J. Li W. Hanson C. Guo J. Zhang S. J. Biol. Chem. 2010; 285: 27259-27264Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). However, the cellular machinery for hERG endocytic degradation is largely unknown. In particular, although ubiquitin (Ub) is found to be involved in the internalization of cell surface hERG channels (6Guo J. Massaeli H. Xu J. Jia Z. Wigle J.T. Mesaeli N. Zhang S. J. Clin. Investig. 2009; 119: 2745-2757Crossref PubMed Scopus (123) Google Scholar, 7Massaeli H. Guo J. Xu J. Zhang S. Circ. Res. 2010; 106: 1072-1082Crossref PubMed Scopus (36) Google Scholar), the nature of Ub-hERG interactions is not known.Ub is a protein with a highly conserved sequence of 76 amino acids found in every eukaryotic cell. The covalent binding of Ub to target proteins is known as ubiquitination, a process well known for labeling proteins for degradation (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 10Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar). Appendage of a single Ub moiety leads to monoubiquitination, whereas attachment of a chain of Ub leads to polyubiquitination. Although polyubiquitination targets proteins for proteasomal degradation (11Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), monoubiquitination targets membrane proteins for internalization and lysosomal degradation. As an internalization signal of membrane proteins, monoubiquitination is well described in yeast (12Galan J.M. Haguenauer-Tsapis R. EMBO J. 1997; 16: 5847-5854Crossref PubMed Scopus (320) Google Scholar, 13Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 14Lucero P. Peñalver E. Vela L. Lagunas R. J. Bacteriol. 2000; 182: 241-243Crossref PubMed Scopus (32) Google Scholar). In mammals, evidence of this connection is also accumulating (9Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 15Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (652) Google Scholar, 16Patrick G.N. Bingol B. Weld H.A. Schuman E.M. Curr. Biol. 2003; 13: 2073-2081Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 17Gupta-Rossi N. Six E. LeBail O. Logeat F. Chastagner P. Olry A. Israël A. Brou C. J. Cell Biol. 2004; 166: 73-83Crossref PubMed Scopus (181) Google Scholar, 18d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (201) Google Scholar, 19Sigismund S. Woelk T. Puri C. Maspero E. Tacchetti C. Transidico P. Di Fiore P.P. Polo S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2760-2765Crossref PubMed Scopus (653) Google Scholar, 20Chen H. De Camilli P. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2766-2771Crossref PubMed Scopus (122) Google Scholar, 21Leithe E. Kjenseth A. Sirnes S. Stenmark H. Brech A. Rivedal E. J. Cell Sci. 2009; 122: 3883-3893Crossref PubMed Scopus (73) Google Scholar, 22Lin D.H. Yue P. Pan C.Y. Sun P. Zhang X. Han Z. Roos M. Caplan M. Giebisch G. Wang W.H. J. Biol. Chem. 2009; 284: 29614-29624Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).In the present study, using biochemical and electrophysiological approaches, we demonstrated, for the first time, that under low K+ conditions monoubiquitination of cell surface hERG channels directs the channel internalization and degradation through the multivesicular body (MVB) pathway, and Ub is released from the internalized channel prior to the channel entry into lysosomes for further degradation.