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• W3134658515 abstract "Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels. Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels. The ether-à-go-go family of voltage-gated K+ channels comprises three gene subfamilies: eag (KV10), erg (eag-related gene) (KV11), and elk (eag-like K+ channel) (KV12) (1Warmke J.W. Ganetzky B. A family of potassium channel genes related to Eag in Drosophila and mammals.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (846) Google Scholar). In mammals, Eag encodes neuron-specific K+ channels that are expressed in various regions of the brain and make significant contributions to subthreshold K+ currents; nonetheless, the specific physiological roles of Eag K+ channels are not yet fully understood (2Saganich M.J. Machado E. Rudy B. Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain.J. Neurosci. 2001; 21: 4609-4624Crossref PubMed Google Scholar, 3Saganich M.J. Vega-Saenz de Miera E. Nadal M.S. Baker H. Coetzee W.A. Rudy B. Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex.J. Neurosci. 1999; 19: 10789-10802Crossref PubMed Google Scholar, 4Ludwig J. Terlau H. Wunder F. Bruggemann A. Pardo L.A. Marquardt A. Stuhmer W. Pongs O. Functional expression of a rat homologue of the voltage gated either a go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart.EMBO J. 1994; 13: 4451-4458Crossref PubMed Scopus (147) Google Scholar, 5Ludwig J. Weseloh R. Karschin C. Liu Q. Netzer R. Engeland B. Stansfeld C. Pongs O. Cloning and functional expression of rat Eag2, a new member of the ether-a-go-go family of potassium channels and comparison of its distribution with that of Eag1.Mol. Cell. Neurosci. 2000; 16: 59-70Crossref PubMed Scopus (66) Google Scholar). The mammalian Eag subfamily encompasses two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2) (3Saganich M.J. Vega-Saenz de Miera E. Nadal M.S. Baker H. Coetzee W.A. Rudy B. Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex.J. Neurosci. 1999; 19: 10789-10802Crossref PubMed Google Scholar, 5Ludwig J. Weseloh R. Karschin C. Liu Q. Netzer R. Engeland B. Stansfeld C. Pongs O. Cloning and functional expression of rat Eag2, a new member of the ether-a-go-go family of potassium channels and comparison of its distribution with that of Eag1.Mol. Cell. Neurosci. 2000; 16: 59-70Crossref PubMed Scopus (66) Google Scholar, 6Schonherr R. Gessner G. Lober K. Heinemann S.H. Functional distinction of human EAG1 and EAG2 potassium channels.FEBS Lett. 2002; 514: 204-208Crossref PubMed Scopus (57) Google Scholar, 7Frings S. Brull N. Dzeja C. Angele A. Hagen V. Kaupp U.B. Baumann A. Characterization of ether-a-go-go channels present in photoreceptors reveals similarity to IKx, a K+ current in rod inner segments.J. Gen. Physiol. 1998; 111: 583-599Crossref PubMed Scopus (72) Google Scholar, 8Ju M. Wray D. Molecular identification and characterisation of the human Eag2 potassium channel.FEBS Lett. 2002; 524: 204-210Crossref PubMed Scopus (54) Google Scholar). Both in situ hybridization and real-time PCR analyses support the notion that Eag1 and Eag2 are highly expressed in a wide variety of different rat brain areas (2Saganich M.J. Machado E. Rudy B. Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain.J. Neurosci. 2001; 21: 4609-4624Crossref PubMed Google Scholar, 5Ludwig J. Weseloh R. Karschin C. Liu Q. Netzer R. Engeland B. Stansfeld C. Pongs O. Cloning and functional expression of rat Eag2, a new member of the ether-a-go-go family of potassium channels and comparison of its distribution with that of Eag1.Mol. Cell. Neurosci. 2000; 16: 59-70Crossref PubMed Scopus (66) Google Scholar, 9Martin S. Lino de Oliveira C. Mello de Queiroz F. Pardo L.A. Stuhmer W. Del Bel E. Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain.Neuroscience. 2008; 155: 833-844Crossref PubMed Scopus (46) Google Scholar, 10Martin S. Lino-de-Oliveira C. Joca S.R. Weffort de Oliveira R. Echeverry M.B. Da Silva C.A. Pardo L. Stuhmer W. Bel E.D. Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats.Genes Brain Behav. 2010; 9: 918-924Crossref PubMed Scopus (7) Google Scholar), implying that the two mammalian Eag isoforms may participate in certain essential functions in the brain. In Drosophila, mutations in the eag gene manifest a hyperexcitable phenotype in which fly legs shake extensively under ether anesthesia (11Ganetzky B. Wu C.F. Neurogenetic analysis of potassium currents in Drosophila: Synergistic effects on neuromuscular transmission in double mutants.J. Neurogenet. 1983; 1: 17-28Crossref PubMed Scopus (137) Google Scholar). Knockout of the Eag1 channel function in mice, however, fails to result in any marked phenotype other than a modest synaptic hyperactivity (12Ufartes R. Schneider T. Mortensen L.S. de Juan Romero C. Hentrich K. Knoetgen H. Beilinson V. Moebius W. Tarabykin V. Alves F. Pardo L.A. Rawlins J.N. Stuehmer W. Behavioural and functional characterization of Kv10.1 (Eag1) knockout mice.Hum. Mol. Genet. 2013; 22: 2247-2262Crossref PubMed Scopus (37) Google Scholar). Interestingly, knockdown of Eag1 in zebrafish reveals a severe disruption in the development of the central nervous system (13Stengel R. Rivera-Milla E. Sahoo N. Ebert C. Bollig F. Heinemann S.H. Schonherr R. Englert C. Kcnh1 voltage-gated potassium channels are essential for early zebrafish development.J. Biol. Chem. 2012; 287: 35565-35575Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Consistent with the observation in zebrafish, mutations in the human gene encoding Eag1 (KCNH1) were recently reported in individuals with two congenital neurodevelopmental disorders: Temple–Baraitser syndrome (TMBTS) (14Simons C. Rash L.D. Crawford J. Ma L. Cristofori-Armstrong B. Miller D. Ru K. Baillie G.J. Alanay Y. Jacquinet A. Debray F.G. Verloes A. Shen J. Yesil G. Guler S. et al.Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.Nat. Genet. 2015; 47: 73-77Crossref PubMed Scopus (84) Google Scholar) and Zimmermann–Laband syndrome (ZLS) (15Bramswig N.C. Ockeloen C.W. Czeschik J.C. van Essen A.J. Pfundt R. Smeitink J. Poll-The B.T. Engels H. Strom T.M. Wieczorek D. Kleefstra T. Ludecke H.J. 'Splitting versus lumping': Temple-Baraitser and Zimmermann-Laband syndromes.Hum. Genet. 2015; 134: 1089-1097Crossref PubMed Scopus (18) Google Scholar, 16Kortum F. Caputo V. Bauer C.K. Stella L. Ciolfi A. Alawi M. Bocchinfuso G. Flex E. Paolacci S. Dentici M.L. Grammatico P. Korenke G.C. Leuzzi V. Mowat D. Nair L.D. et al.Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome.Nat. Genet. 2015; 47: 661-667Crossref PubMed Scopus (122) Google Scholar). TMBTS is characterized by intellectual disability, epilepsy, dysmorphic facial features, and broad thumbs and great toes with absent/hypoplastic nails; whereas ZLS is characterized by facial dysmorphism including coarsening of the face and a large nose, gingival enlargement, intellectual disability, hypoplasia of terminal phalanges and nails, and hypertrichosis. This correlation between KCNH1 mutations and the clinical presentation of TMBTS/ZLS highlights a potentially important role of Eag1 during the development of the brain. We have previously demonstrated that a ZLS-causing mutant Eag1 channel (G469R) is associated with significant reduction in total protein level (17Hsu P.H. Ma Y.T. Fang Y.C. Huang J.J. Gan Y.L. Chang P.T. Jow G.M. Tang C.Y. Jeng C.J. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.Sci. Rep. 2017; 7: 40825Crossref PubMed Scopus (8) Google Scholar), suggesting the presence of a disrupted Eag1 protein homeostasis. Protein homeostasis (also known as proteostasis) refers to the property of a subcellular system that regulates and maintains a stable balance of protein synthesis and degradation (18Douglas P.M. Dillin A. Protein homeostasis and aging in neurodegeneration.J. Cell Biol. 2010; 190: 719-729Crossref PubMed Scopus (238) Google Scholar). Protein homeostatic regulation plays a critical role in determining the optimal expression level of endogenous K+ channels in neurons, which in turn may set the network excitability in a wide variety of different neural circuits in the brain (19Misonou H. Homeostatic regulation of neuronal excitability by K(+) channels in normal and diseased brains.Neuroscientist. 2010; 16: 51-64Crossref PubMed Scopus (46) Google Scholar). Very little is known about the molecular regulation of Eag1 protein homeostasis. Previously, we reported that the E3 ubiquitin ligase cullin 7 (CUL7) promotes proteasomal and lysosomal degradation of endoplasmic reticulum (ER)- and plasma membrane-localized Eag1 proteins, respectively (17Hsu P.H. Ma Y.T. Fang Y.C. Huang J.J. Gan Y.L. Chang P.T. Jow G.M. Tang C.Y. Jeng C.J. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.Sci. Rep. 2017; 7: 40825Crossref PubMed Scopus (8) Google Scholar). In this study, we present the identification of another novel binding partner of Eag1, the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1; also known as RNF61). The results from our biochemical and biophysical analyses are consistent with the idea that MKRN1 contributes to protein quality control of Eag1 K+ channels, as well as ER-associated degradation of disease-causing misfolded Eag1 proteins. To search for the protein machinery responsible for homeostatic regulation of the Eag1 K+ channel, we carried out yeast two-hybrid screening of a rat brain cDNA library by using the cytoplasmic carboxyl (C)-terminal region of rat Eag1 as the bait. Two of the positive clones isolated by the screening correspond to MKRN1, which belongs to the RING E3 ubiquitin ligase family and contains both the RING-finger E2-binding domain and the substrate-binding domain in the same protein (20Gray T.A. Hernandez L. Carey A.H. Schaldach M.A. Smithwick M.J. Rus K. Marshall Graves J.A. Stewart C.L. Nicholls R.D. The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system.Genomics. 2000; 66: 76-86Crossref PubMed Scopus (77) Google Scholar). In addition to regulating protein homeostasis, MKRN1 may also interact with RNA-binding proteins and contribute to mRNA quality control (21Cassar P.A. Carpenedo R.L. Samavarchi-Tehrani P. Olsen J.B. Park C.J. Chang W.Y. Chen Z. Choey C. Delaney S. Guo H. Guo H. Tanner R.M. Perkins T.J. Tenenbaum S.A. Emili A. et al.Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.EMBO Rep. 2015; 16: 1334-1357Crossref PubMed Scopus (18) Google Scholar, 22Hildebrandt A. Bruggemann M. Ruckle C. Boerner S. Heidelberger J.B. Busch A. Hanel H. Voigt A. Mockel M.M. Ebersberger S. Scholz A. Dold A. Schmid T. Ebersberger I. Roignant J.Y. et al.The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation.Genome Biol. 2019; 20: 216Crossref PubMed Scopus (14) Google Scholar). There are two MKRN1 isoforms, the full-length, long isoform (isoform 1; 482 amino acids in human; 481 amino acids in rat) and the C-terminal truncated, short isoform (MKRN1-S; isoform 2; 329 amino acids in both human and rat), both of which are abundantly expressed in the brain (20Gray T.A. Hernandez L. Carey A.H. Schaldach M.A. Smithwick M.J. Rus K. Marshall Graves J.A. Stewart C.L. Nicholls R.D. The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system.Genomics. 2000; 66: 76-86Crossref PubMed Scopus (77) Google Scholar, 23Omwancha J. Zhou X.F. Chen S.Y. Baslan T. Fisher C.J. Zheng Z. Cai C. Shemshedini L. Makorin RING finger protein 1 (MKRN1) has negative and positive effects on RNA polymerase II-dependent transcription.Endocrine. 2006; 29: 363-373Crossref PubMed Scopus (26) Google Scholar, 24Miroci H. Schob C. Kindler S. Olschlager-Schutt J. Fehr S. Jungenitz T. Schwarzacher S.W. Bagni C. Mohr E. Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells.J. Biol. Chem. 2012; 287: 1322-1334Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Importantly, only the long isoform of MKRN1 contains the complete structural domains required for the E3 ubiquitin ligase function (24Miroci H. Schob C. Kindler S. Olschlager-Schutt J. Fehr S. Jungenitz T. Schwarzacher S.W. Bagni C. Mohr E. Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells.J. Biol. Chem. 2012; 287: 1322-1334Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The protein sequences of the two rat MKRN1 clones isolated by yeast two-hybrid screening correspond to the partial segments (amino acids 142–481 and 355–481) of the long isoform and are virtually absent in the short isoform, suggesting that Eag1 may mainly interact with the MKRN1 long isoform. To address this possibility, we went on to perform the co-immunoprecipitation experiment. Rat Eag1 was coexpressed with either Myc-tagged rat MKRN1 long isoform (Myc-MKRN1) or Myc-tagged rat MKRN1 short isoform (Myc-MKRN1-S) in HEK293T cells. By employing both the anti-Myc and the anti-Eag1 antibodies for immunoprecipitation, we demonstrate in Figure 1A that only the long isoform of MKRN1, but not the short isoform, was co-immunoprecipitated with Eag1. We also generated a GST fusion protein encoding the C-terminal region (amino acids 355–481) of the MKRN1 long isoform (GST-MKRN1-C) and performed GST pull-down assay using rat brain lysates. As shown in Figure 1B, endogenous Eag1 protein in the brain lysates was readily precipitated by GST-MKRN1-C. Additionally, endogenous MKRN1 and Eag1 coexist in the same protein complex in neurons (Fig. S1). The direct interaction between Eag1 and MKRN1 long isoform, but not the short isoform, in HEK293T cells was further validated by the in situ proximity ligation assay (PLA) (Fig. 1C), which detects immunofluorescence signals generated from a pair of oligonucleotide-linked antibodies recognizing two proteins that are in close proximity (<40 nm) (25Fredriksson S. Gullberg M. Jarvius J. Olsson C. Pietras K. Gustafsdottir S.M. Ostman A. Landegren U. Protein detection using proximity-dependent DNA ligation assays.Nat. Biotechnol. 2002; 20: 473-477Crossref PubMed Scopus (975) Google Scholar, 26Soderberg O. Leuchowius K.J. Gullberg M. Jarvius M. Weibrecht I. Larsson L.G. Landegren U. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.Methods. 2008; 45: 227-232Crossref PubMed Scopus (384) Google Scholar, 27Herbert L.M. Nitta C.H. Yellowhair T.R. Browning C. Gonzalez Bosc L.V. Resta T.C. Jernigan N.L. PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells.Am. J. Physiol. Cell Physiol. 2016; 310: C390-400Crossref PubMed Scopus (10) Google Scholar). Together these observations indicate that Eag1 interacts primarily with the C-terminal region of the long isoform of MKRN1. The bait sequence used for screening the rat brain cDNA library corresponds to the cytoplasmic C-terminal region (amino acids 493–962) of rat Eag1, suggesting that MKRN1 probably interacts with the C-terminal region of Eag1. To test this hypothesis, we generated GST fusion proteins encoding either the amino (N)-terminal (GST-N207) or the C-terminal (GST-C0) region of rat Eag1 (Fig. 1D). Indeed, MKRN1 was effectively pulled down by the GST-Eag1 fusion protein GST-C0, but not by GST-N207. To further locate the MKRN1-interacting domain in Eag1, we generated three additional GST fusion proteins containing different Eag1 C-terminal domains: GST-C1 (amino acids 493–724), GST-C2 (amino acids 723–848), and GST-C3 (amino acids 835–962) (Fig. 1D). MKRN1 was efficiently pulled down by GST-C1, but not by GST-C2 and GST-C3, indicating that the proximal C-terminal region of Eag1 harbors the major MKRN1-interacting domain. The proximal C-terminal region of Eag1 contains a C-linker domain and a cyclic nucleotide-binding homology domain (CNBHD). We therefore made two additional GST fusion proteins that encode either the C-linker (GST-C1A: amino acid 493–560) or the CNBHD (GST-C1B: amino acids 561–722) region. As depicted in Figure 1D, MKRN1 preferentially binds to GST-C1B, implying that MKRN1 may directly interact with the Eag1 CNBHD. Upon close inspection of the PLA immunofluorescence signals of Eag1 and MKRN1 long isoform (Fig. 1C), which probably reflect the direct interaction of the two macromolecules, we noticed that the majority of the signals was localized at the cytoplasmic perinuclear region. Since properly folded, mature Eag1 proteins are generally considered plasma membrane ion channels, the foregoing PLA images appear to imply that MKRN1 mainly interacts with immature Eag1 proteins that either have yet to or even fail to reach the plasma membrane. To address this interesting possibility, we studied the effect of MKRN1 on Eag1 protein maturation in HEK293T cells. Eag1 harbors two consensus asparagine (N)-linked glycosylation sites (N388 and N406) (28Napp J. Monje F. Stuhmer W. Pardo L.A. Glycosylation of Eag1 (Kv10.1) potassium channels: Intracellular trafficking and functional consequences.J. Biol. Chem. 2005; 280: 29506-29512Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Importantly, the electrophoretic mobility pattern of Eag1 proteins on an immunoblot typically manifests as two major protein bands: i) a slower (high-molecular-weight) band “a” that corresponds to mature, full-glycosylated Eag1 proteins localized at the plasma membrane, and ii) a faster (low-molecular-weight) band “b” that represents immature, core-glycosylated Eag1 proteins at the ER (Fig. 2A) (17Hsu P.H. Ma Y.T. Fang Y.C. Huang J.J. Gan Y.L. Chang P.T. Jow G.M. Tang C.Y. Jeng C.J. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.Sci. Rep. 2017; 7: 40825Crossref PubMed Scopus (8) Google Scholar). By coexpressing Eag1 with MKRN1, we noticed the presence of another Eag1-associated low-molecular-weight protein band “c” (Fig. 2A). In contrast, no significant band c was detected when Eag1 was coexpressed with the noninteracting MKRN1-S (Fig. 2A). Since the apparent electrophoretic mobility of the MKRN1-induced Eag1 band c is significantly faster than that of immature, core-glycosylated Eag1 proteins (band b), it is possible that band c may correspond to nascent, nonglycosylated Eag1 proteins. To address this hypothesis, we employed an Eag1 mutant with the two consensus glycosylation asparagine residues mutated into glutamine (N388Q and N406Q; Eag1-QQ). Figure 2B shows that removing all N-linked oligosaccharides of Eag1 (both bands a and b) with the amidase PNGase F generates low-molecular-weight protein that appears to comigrate with the nonglycosylated Eag1-QQ mutant. Furthermore, cleaving high mannose oligosaccharides from immature, core-glycosylated Eag1 (band b) with Endo H also results in the presence of the Eag1-QQ-like, low-molecular-weight protein band (Fig. 2B). Mass spectrometry analyses of bands a, b, and c, as well as the single, deglycosylated band in response to PNGase F treatment, reveal that they indeed represent Eag1 proteins of distinct apparent molecular weights (Fig. S2; Table S1). Most importantly, all the above-mentioned nonglycosylated Eag1 protein bands share virtually identical electrophoretic mobility pattern with the MKRN1-induced Eag1 band c, consistent with the idea that band c represents nascent, nonglycosylated Eag1 proteins. How can MKRN1 enhance the presence of nascent, nonglycosylated Eag1 proteins? One possibility is that MKRN1 may promote the deglycosylation of core-glycosylated Eag1 and/or prevent the glycosylation of nascent Eag1. If this inference is true, MKRN1 would be expected to display a differential binding affinity between immature Eag1 (bands b and c) and mature Eag1 (band a). Indeed, Figure 2C illustrates that, compared with their mature counterpart, immature Eag1 proteins were more efficiently co-immunoprecipitated with MKRN1. In contrast, no discernible difference in the co-immunoprecipitation efficiency with MKRN1 was detected between immature Eag1 proteins (bands b and c) and the nonglycosylated Eag1-QQ mutant (Fig. 2D). Taken together, the foregoing results support the notion that MKRN1 preferentially interacts with immature Eag1 proteins that are probably located at the cytoplasm, such as the ER or the cis-Golgi apparatus. Next we asked whether MKRN1 may regulate protein homeostasis of Eag1 K+ channels. Upon siRNA or shRNA knockdown of endogenous MKRN1 expression in HEK293T cells, we observed a sizeable upregulation of total Eag1 protein level (Fig. 3, A and B; Fig. S3). A similar Eag1 upregulation effect was also detected when we knocked down endogenous MKRN1 expression in cultured cortical neurons (Fig. 3C). Conversely, overexpression of MKRN1, but not the short isoform (MKRN1-S) or a catalytically inactive MKRN1 construct with a point mutation in the E3 ligase domain (MKRN1-H307E) (29Lee E.W. Lee M.S. Camus S. Ghim J. Yang M.R. Oh W. Ha N.C. Lane D.P. Song J. Differential regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and apoptosis.EMBO J. 2009; 28: 2100-2113Crossref PubMed Scopus (107) Google Scholar), results in a substantial reduction of both immature (bands b and c) and mature (band a) Eag1 proteins in HEK293T cells (Fig. 3D). Similarly, only MKRN1, but not MKRN1-S and MKRN1-H307E, impairs the functional expression of Eag1 K+ currents in HEK293T cells (Fig. 3E). MKRN1 fails to discernibly affect the biophysical property of Eag1 K+ channels, consistent with the idea that the observed reduction in membrane K+ conductance primarily reflects a disruption of Eag1 protein homeostasis by MKRN1. Moreover, in agreement with the aforementioned interaction of MKRN1 with the nonglycosylated Eag1-QQ mutant, coexpression with MKRN1 considerably decreases Eag1-QQ protein level (Fig. S4A). We then address the mechanism underlying the downregulation of Eag1 protein level by MKRN1. Semiquantitative RT-PCR analyses reveal that MKRN1 does not detectably affect the mRNA level of Eag1 (Fig. S5). This observation appears to rule out a potential transcriptional effect of MKRN1 on Eag1. Given its known role as a member of the RING finger E3 ubiquitin ligase superfamily (20Gray T.A. Hernandez L. Carey A.H. Schaldach M.A. Smithwick M.J. Rus K. Marshall Graves J.A. Stewart C.L. Nicholls R.D. The ancient source of a distinct gene family encoding proteins featuring RING and C(3)H zinc-finger motifs with abundant expression in developing brain and nervous system.Genomics. 2000; 66: 76-86Crossref PubMed Scopus (77) Google Scholar), we also explored the possibility that MKRN1 may affect Eag1 protein homeostasis by catalyzing the ubiquitination of the K+ channel. Figure 4A illustrates that, in response to coexpression with HA-tagged ubiquitin (HA-Ub), basal Eag1 ubiquitination signal manifests as a faint, diffuse Eag1 protein smear conjugated with HA-Ub, consistent with the presence of Eag1 polyubiquitination in HEK293T cells. Importantly, overexpression of MKRN1 leads to a prominent enhancement of Eag1 polyubiquitination (Fig. 4A). Moreover, MKRN1 promotes polyubiquitination of the nonglycosylated Eag1-QQ mutant (Fig. S4B), further supporting the notion that glycosylation is not required for Eag1 regulation by MKRN1. In contrast, overexpression of either the short isoform MKRN1-S or the catalytically inactive mutant MKRN1-H307E fails to exert comparable ubiquitination-enhancing effect on Eag1 (Fig. 4B). Together these results suggest that MKRN1 effectively promotes Eag1 polyubiquitination, which leads to substantial downregulation of Eag1 protein level. To ascertain the detailed mechanism of Eag1 downregulation by MKRN1, we went on to study the effect of two distinct types of protein degradation blockers, the lysosome inhibitor chloroquine (CQ) and the proteasome inhibitor MG132. Figure 5A demonstrates that, for both mature (band a) and immature (bands b and c) Eag1, the extent of protein downregulation by MKRN1 remains virtually the same in the absence or presence of CQ, suggesting against a mechanistic role of lysosomal degradation. Application of MG132 does not notably affect the downregulation of mature Eag1 by MKRN1 either (Fig. 5B). Nevertheless, MG132 treatment effectively abolishes the suppression effect of MKRN1 on the protein level of immature Eag1 (Fig. 5B). The selective rescue effect of MG132 on immature but not mature Eag1 appears to imply that MKRN1 efficiently promotes proteasomal degradation of immature Eag1. Furthermore, although MG132 markedly rescues MKRN1-promoted degradation of immature Eag1, the majority of the rescued immature Eag1 bands b and c may be substantially misfolded and is thus prevented from protein maturation into the mature Eag1 band a. So far our data are consistent with the idea that MKRN1 preferentially interacts with and promotes degradation of immature Eag1, which suggests that both MKRN1 and immature Eag1 may be localized at the ER membrane. To directly address this hypothesis, we examined the subcellular localization of Eag1 and MKRN1 in HEK293T cells. Differential centrifugation analyses indicate that the ER-resident membrane protein calnexin, the plasma membrane protein cadherin, and Eag1 are exclusively present in the membrane fraction (Fig. 5C). Sucrose density gradient analyses further demonstrate that, in the absence of MKRN1, mature (band a) and immature (band b) Eag1 proteins were preferentially detected in the cadherin-enriched plasma membrane and the calnexin-enriched ER membrane fractions, respectively (Fig. 5C). In HEK293T cells, both endogenous and heterologously expressed MKRN1 display notable membrane association as well; moreover, in the presence of MKRN1, both immature Eag1 proteins (bands b and c) and MKRN1 are present in the calnexin-enriched ER membrane fraction (" @default.
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