Ubiquitylation of the gap junction protein connexin-43 signals its … · 2009. 11. 4. · (Fig. 1). The two upper bands (Cx43-P1 and Cx43-P2) represent Cx43 organized in gap junctions

3883Research Article

IntroductionGap junctions are arrays of intercellular channels that enableneighbouring cells to communicate directly by exchanging ions,signalling molecules and small metabolites (Saez et al., 2003). Gapjunction channels are composed of two channel structures calledconnexons, each of which are contributed by the two adjacent cells.Connexons consist of six connexin proteins. There are 21 knownmembers of the connexin protein family in the human genome, ofwhich the best-studied isoform is connexin-43 (Cx43; also knownas Gja1) (Sohl and Willecke, 2003). Connexins play important rolesin the regulation of cell growth and tissue homeostasis, anddysfunctional intercellular communication via gap junctions hasbeen implicated as a causative factor in heart failure,neuropathology, deafness, skin disorders and cataracts (Wei et al.,2004). There is also significant evidence that loss of gap junctionalcommunication is an important step in carcinogenesis (Leithe etal., 2006b; Mesnil et al., 2005).

Gap junctions are dynamic plasma membrane domains. Newlysynthesized connexons are continually added to the edges ofexisting gap junctions and dock with connexons in the adjacent cellsto form functional intercellular channels (Gaietta et al., 2002; Laufet al., 2002). Following their assembly into gap junctions, Cx43moves toward the centre of the gap junction, where it is removedby endocytosis (Gaietta et al., 2002; Lauf et al., 2002). Cx43 hasa high turnover rate in most tissues, with a half-life of 1.5-5 hours(Fallon and Goodenough, 1981; Laird et al., 1991). Cx43 is knownto be tightly regulated by phosphorylation (Solan and Lampe, 2009).

Among the kinases involved in phosphorylation of Cx43 areprotein kinase C (PKC) and mitogen-activated protein (MAP) kinase(Lampe et al., 2000; Warn-Cramer et al., 1998). Modulation ofconnexin degradation has been suggested to be an importantmechanism by which cells regulate the level of gap junctionalintercellular communication (Berthoud et al., 2004; Laird, 2005;Musil et al., 2000; Thomas et al., 2003). However, the molecularmachinery involved in mediating connexin degradation hasremained poorly understood.

During endocytosis of gap junctions, both membranes of thejunction are internalized into one of the adjacent cells and therebyform a double-membrane vacuole called an annular gap junctionor connexosome (Jordan et al., 2001; Larsen and Hai, 1978; Leitheet al., 2006a; Nickel et al., 2008; Piehl et al., 2007). Followinginternalization of gap junctions, connexins are degraded inlysosomes (Laing et al., 1997; Leithe and Rivedal, 2004b; Leitheet al., 2006a; Naus et al., 1993; Qin et al., 2003; VanSlyke et al.,2000; Vaughan and Lasater, 1990). Based on immunoelectronmicroscopy studies, we have previously proposed that theinternalized, annular gap junction undergoes a maturation processfrom a double membrane vacuole to a multivesicular endosome witha single limiting membrane (Leithe et al., 2006a). This processingof the annular gap junction was found to be associated withtrafficking of Cx43 to early endosomes, prior to its degradation inlysosomes (Leithe et al., 2006a). The observation that the gapjunction double membrane is processed into two single membranesafter its internalization implies that the gap junction channels undock

Gap junctions are dynamic plasma membrane domains, andtheir protein constituents, the connexins, have a high turnoverrate in most tissue types. However, the molecular mechanismsinvolved in degradation of gap junctions have remained largelyunknown. Here, we show that ubiquitin is strongly relocalizedto connexin-43 (Cx43; also known as Gja1) gap junction plaquesin response to activation of protein kinase C. Cx43 remainedubiquitylated during its transition to a Triton X-100-solublestate and along its trafficking to early endosomes. Followinginternalization, Cx43 partly colocalized with the ubiquitin-binding proteins Hrs (hepatocyte growth factor-regulatedtyrosine kinase substrate; also known as Hgs) and Tsg101(tumor susceptibility gene 101). Depletion of Hrs or Tsg101 bysmall interfering RNA abrogated trafficking of Cx43 from earlyendosomes to lysosomes. Under these conditions, Cx43 was able

to undergo dephosphorylation and deubiquitylation, locate tothe plasma membrane and form functional gap junctions.Simultaneous depletion of Hrs and Tsg101 caused accumulationof a phosphorylated and ubiquitylated subpopulation of Cx43in early endosomes and in hybrid organelles between partlydegraded annular gap junctions and endosomes. Collectively,these data reveal a central role of early endosomes in sortingof ubiquitylated Cx43, and identify Hrs and Tsg101 as crucialregulators of trafficking of Cx43 to lysosomes.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/21/3883/DC1

Key words: Ubiquitin, Gap junctions, Connexin-43, Hrs, Tsg101,Early endosome, Endocytosis

Summary

Ubiquitylation of the gap junction protein connexin-43signals its trafficking from early endosomes tolysosomes in a process mediated by Hrs and Tsg101Edward Leithe*, Ane Kjenseth, Solveig Sirnes, Harald Stenmark, Andreas Brech and Edgar RivedalCentre for Cancer Biomedicine, Faculty of Medicine, University of Oslo and Institute for Cancer Research, the Norwegian Radium Hospital, OsloUniversity Hospital, Montebello, Oslo, Norway*Author for correspondence ([email protected])

Accepted 13 August 2009Journal of Cell Science 122, 3883-3893 Published by The Company of Biologists 2009doi:10.1242/jcs.053801

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during or shortly after internalization. Thus, in this scenario for Cx43degradation, most Cx43 localized in early endosomes is expectedto exist as undocked connexons. The early endosome is a majorsorting station for proteins internalized from the plasma membrane(Raiborg and Stenmark, 2009). After entering the early endosome,endocytosed proteins can be trafficked further along the degradationpathway to the lysosome, be recycled to the plasma membrane, orbe transported to the trans-Golgi network. By orchestrating thetrafficking of endocytosed proteins, early endosomes play importantroles in regulating the levels of proteins at the plasma membrane(Gould and Lippincott-Schwartz, 2009). However, the functionalimportance of the early endosome in the formation and degradationof gap junctions has not been investigated.

As early endosomes mature into late endosomes, proteins thatare destined for lysosomal degradation are incorporated intointraluminal vesicles that bud from the limiting membrane of theendosome (Raiborg and Stenmark, 2009). Subsequently, fusionbetween late endosomes and lysosomes causes proteolyticdegradation of the intravacuolar vesicles and their proteins.Transport of proteins into multivesicular endosomes requirespositive sorting signals present at the cytosolic domains of theproteins. Conjugation of ubiquitin is the best-known sorting signalfor lysosomal trafficking of endocytosed growth factor receptors(Raiborg and Stenmark, 2009). Ubiquitylated growth factorreceptors are recognized by the ubiquitin-binding protein Hrs(hepatocyte growth factor-regulated tyrosine kinase substrate; alsoknown as Hgs), which together with STAM (signal-transducingadaptor molecule) and Eps15b forms a complex at endosomesreferred to as ESCRT (endosomal sorting complex required fortransport)-0 (Bache et al., 2003; Bilodeau et al., 2002; Hirano etal., 2006; Raiborg et al., 2002; Roxrud et al., 2008). Subsequently,the ubiquitylated receptor is thought to be transferred to theubiquitin-binding protein Tsg101 (tumor susceptibility gene 101),which is part of a heterotrimeric complex called ESCRT-I (Babstet al., 2000; Bishop and Woodman, 2001; Bishop et al., 2002; Luet al., 2003). The cargo protein is then transported to the proteincomplexes ESCRT-II and -III, before its incorporation intointraluminal vesicles (Piper and Katzmann, 2007). Prior to thetransport of the receptor into the lumen of the endosome, it isdeubiquitylated by isopeptidases recruited by components in theESCRT-III complex, enabling reuse of ubiquitin (Kato et al., 2000;McCullough et al., 2006; Tanaka et al., 1999). Endocytosedreceptors that do not undergo ubiquitylation are not sorted to thelysosome but are instead recycled from the early endosome to theplasma membrane (Raiborg and Stenmark, 2009).

Several lines of evidence indicate that ubiquitin plays a centralrole in the regulation of Cx43 degradation. Firstly, Cx43 is able toundergo ubiquitylation, and inactivation of the ubiquitin-activatingenzyme E1 is associated with increased levels of Cx43 protein (Laingand Beyer, 1995). Secondly, the E3 ubiquitin ligase Nedd4 binds toCx43 and regulates the level of gap junctions at the plasma membrane(Leykauf et al., 2006). Thirdly, ubiquitylation of Cx43 is stronglyinduced in response to activation of MAP kinase or PKC (Leithe andRivedal, 2004a; Leithe and Rivedal, 2004b). However, the functionalrole of ubiquitin in the degradation of gap junctions remains unknown.In the present study, we show that endocytosis of Cx43 is associatedwith a strong relocalization of ubiquitin to gap junction plaques. Weprovide evidence that Cx43 remains ubiquitylated along its traffickingto early endosomes. The data suggest that early endosomes have anessential role in mediating trafficking of Cx43 to lysosomes, in aprocess mediated by Hrs and Tsg101.

ResultsUbiquitin is recruited to Cx43 gap junction plaques in responseto PKC activationUbiquitylation of Cx43 in IAR20 cells is efficiently induced byexposure to epidermal growth factor (EGF) or the PKC activator12-O-tetradecanoylphorbol 13-acetate (TPA) (Leithe and Rivedal,2004a; Leithe and Rivedal, 2004b). We have previously shown thatEGF- and TPA-induced endocytosis of Cx43 gap junctions isassociated with loss of the detergent resistance of Cx43 (Sirnes etal., 2008). This loss of the Cx43 detergent resistance was suggestedto reflect the separation of the gap junction double membrane duringendocytosis, as observed by immunoelectron microscopy (Leitheet al., 2006a). As a first approach to elucidate the role of ubiquitinin the endocytosis of gap junctions, we considered it important todetermine the detergent resistance of the ubiquitylated Cx43 pool.Cx43 from IAR20 cells, forms three distinct bands on SDS-PAGE(Fig. 1). The two upper bands (Cx43-P1 and Cx43-P2) representCx43 organized in gap junctions and they are Triton X-100 resistantin unstimulated cells (Musil and Goodenough, 1991; Sirnes et al.,2008). As expected, TPA strongly induced transformation of Cx43into a Triton X-100 soluble fraction (Fig. 1). Interestingly, theubiquitylated Cx43 pool was found both in the Triton X-100-insoluble and -soluble fractions. These data suggest thatubiquitylated Cx43 exists both in intact gap junctions (i.e. in a TritonX-100-insoluble state) as well as in gap junctions that are in theprocess of loosing their double membrane structure (i.e. in a TritonX-100-soluble state).

We next aimed to elucidate the association between ubiquitinand Cx43 by confocal immunofluorescence microscopy. For thispurpose, we used the FK2 antibody, which specifically detectsubiquitin conjugated to proteins (Fujimuro et al., 1994). Inagreement with previous findings, most Cx43 in IAR20 cells wasorganized as gap junctions between neighbouring cells (Fig. 2A)(Leithe and Rivedal, 2004a; Leithe and Rivedal, 2004b). Asexpected, ubiquitin was found to localize both in the nucleus andin the cytoplasm (Fig. 2A). Under normal cell growth conditions,ubiquitin was rarely found to colocalize with gap junctions.Importantly, treating the cells with TPA for 15 minutes caused adramatic relocalization of ubiquitin to Cx43 gap junction plaques(Fig. 2B; supplementary material Fig. S1A). Interestingly, the Cx43

Journal of Cell Science 122 (21)

Fig. 1. Ubiquitylated Cx43 exists both in a Triton-X-100-insolubleand -soluble state. IAR20 cells were left untreated or treated with TPA (100ng/ml) for 15 or 30 minutes. Cells were subsequently subjected to a Triton X-100 solubility assay. The total cell lysate fraction (T), the Triton-X-100-solublefraction (S) and the Triton-X-100-insoluble fraction (I) were subjected tocoimmunoprecipitation with anti-Cx43 antibodies. Equal amounts ofimmunoprecipitates were subjected to SDS-PAGE. Ubiquitin was detected bywestern blotting using the P4D1 anti-ubiquitin antibody (upper panel) andCx43 with anti-Cx43 antibodies (lower panel). The three major Cx43 SDS-PAGE bands, called P0, P1 and P2, are indicated.

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3885Ubiquitin in gap junction endocytosis

gap junctions that colocalized with ubiquitin were often observedto be in the process of internalizing from the plasma membraneinto one of the adjacent cells. Internalization of gap junctionsoccurred both in the centre and in the periphery of the gap junctionplaque. Often, only one or two gap junctions in a cell were foundto be in the process of internalizing into the cytosol (Fig. 2C). Inthese cases, the gap junctions that were in the process of internalizingcolocalized with ubiquitin, whereas the other gap junctions did notcolocalize with ubiquitin.

Sometimes, annular gap junctions that had completely detachedfrom the plasma membrane (Fig. 2D) or were in the process ofbeing internalized (supplementary material Fig. S1B) wereobserved. The annular gap junctions were found to colocalize withubiquitin. Notably, however, most gap junctions that were in the

process of internalizing from the plasma membrane did not appearto form annular gap junctions. Instead, the gap junctions thatinternalized showed a more diffuse Cx43 staining compared to gapjunction areas that were not in the process of internalizing (Fig.2B,C). This diffuse Cx43 staining possibly reflects the separationof the gap junction double membrane and loss of the detergentresistance of Cx43, as described above and in previous studies(Leithe et al., 2006a; Sirnes et al., 2008).

The TPA-induced colocalization between ubiquitin and Cx43 gapjunctions was counteracted by the PKC inhibitor GF109203 (Fig.2E), in accordance with our previous finding that TPA-inducedubiquitylation of Cx43 is mediated by PKC (Leithe and Rivedal,2004b). Also the MEK1 inhibitor PD98059 strongly counteractedthe TPA-induced recruitment of ubiquitin to Cx43 gap junctions(supplementary material Fig. S1C), in agreement with our previousobservation that PKC-induced ubiquitylation of Cx43 is partlymediated through the MAP kinase pathway (Leithe and Rivedal,2004b).

Collectively, these results suggest that ubiquitin colocalizes withCx43 gap junction plaques in response to PKC activation, and thatCx43 remains ubiquitylated during and after internalization.

Trafficking of Cx43 from early endosomes to lysosomes isregulated by Hrs and Tsg101In agreement with previous studies, most Cx43 was found to localizein intracellular vesicles following 30 minutes of TPA exposure (Fig.3A) (Leithe and Rivedal, 2004b). Interestingly, these vesicles wereoften found to be ubiquitin positive, supporting the notion that Cx43remains ubiquitylated after internalization. We have previouslyreported that following internalization of Cx43 gap junctions, Cx43

Fig. 2. Activation of PKC induces recruitment of ubiquitin to Cx43 gapjunction plaques. IAR20 cells were left untreated (A), treated with TPA (100ng/ml) for 15 minutes (B-D), or co-incubated with TPA (100 ng/ml) andGF109203 (10M) for 15 minutes (E). Cells were double-stained with anti-Cx43 antibodies and the FK2 anti-ubiquitin antibody as indicated andvisualized using confocal immunofluorescence microscopy. Merged imagesare shown in the right panels, with yellow indicating colocalization. Arrowsindicate part of gap junctions that are in the process of internalizing into one ofthe adjacent cells. These gap junctions often appeared to have a more diffuseCx43 staining than gap junction areas that were not in the process ofinternalizing. Scale bars: 5m.

Fig. 3. Cx43 colocalizes with ubiquitin, Hrs and Tsg101 in intracellularcompartments. IAR20 cells were treated with TPA (100 ng/ml) for 30 minutes,and double stained with anti-Cx43 antibodies and either FK2 anti-ubiquitinantibodies, or anti-Hrs or anti-Tsg101 antibodies. Cells were visualized usingconfocal immunofluorescence microscopy. Merged images are shown in theright panels, with yellow indicating colocalization. Insets show enlarged viewsof representative vesicles, showing colocalization. Scale bars: 5m.

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is trafficked via the early endosome before its degradation inlysosomes (Leithe et al., 2006a). Based on the above data, showingthat Cx43 remained ubiquitylated after its internalization from theplasma membrane, we asked whether ubiquitylation of Cx43 couldplay a role in mediating trafficking of Cx43 from the earlyendosome to the lysosome. To test this hypothesis, we focused onthe ubiquitin-binding proteins Hrs and Tsg101. Both proteins havebeen reported to interact with ubiquitylated growth factor receptorsat the early endosome and are essential for mediating their traffickingto the lysosome (Raiborg and Stenmark, 2009). Interestingly,following 30 minutes of TPA treatment, Cx43 was found to partlycolocalize with Hrs and Tsg101 in intracellular vesicles, asdetermined by confocal microscopy (Fig. 3B,C).

To investigate the functional importance of Hrs and Tsg101 inCx43 degradation, endogenous Hrs and Tsg101 were depleted bysmall interfering RNA (siRNA). Following transfection of IAR20cells with siRNA sequences to Hrs and Tsg101 alone or incombination, the Hrs protein level was reduced by approximately70% compared with the control, whereas Tsg101 expression was

reduced approximately 95%, compared to cells transfected with acontrol siRNA sequence (Fig. 4A). Depletion of Hrs did not causemajor changes in the localization of Cx43 in untreated cells (Fig.4B). Cells in which Tsg101 was depleted appeared to haveincreased Cx43 staining and enlarged gap junctions compared withcontrol cells. However, cells depleted of Tsg101 did not show moreCx43 staining in intracellular vesicles than control cells. Bycontrast, when Hrs and Tsg101 were simultaneously depleted, Cx43was found to localize both in intracellular vesicles and in gapjunctions (Fig. 4B).

We next aimed to elucidate whether Hrs and Tsg101 are involvedin PKC-induced degradation of Cx43. In these experiments, TPA wasco-incubated with cycloheximide, to block new protein synthesis. Asexpected, TPA treatment for 1.5 hours caused a strong loss of Cx43staining in cells transfected with a control siRNA sequence, asdetermined by immunofluorescence microscopy (Fig. 4B). Bycontrast, cells in which Hrs or Tsg101 was depleted showed significantstaining of Cx43 in intracellular vesicles at this time point. Asdetermined by confocal microscopy, these vesicles were often found


Fig. 4. Depletion of Hrs and Tsg101 differentially affect Cx43 gapjunctions under constitutive conditions and in response to TPAtreatment. (A)IAR20 cells were transfected with control siRNA orwith siRNA against Hrs or Tsg101 alone or simultaneously. Celllysates were prepared 48 hours after transfection, and equalamounts of total cell protein were subjected to SDS-PAGE. Hrsand Tsg101 were detected by western blotting, using anti-Hrs oranti-Tsg101 antibodies. The Hrs and Tsg101 band intensities weremeasured using Scion Image. Values shown are the mean ± s.d.of three independent experiments. (B)IAR20 cells weretransfected with control siRNA or with siRNA against Hrs orTsg101 alone or simultaneously. 48 hours after transfection, cellswere co-incubated with TPA (100 ng/ml) and cycloheximide (chx,10M) for 1.5 or 3 hours. Cells were stained with anti-Cx43antibodies and visualized using immunofluorescence microscopy.

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to be positive for the early endosome marker EEA1 (Fig. 5). TheTPA-induced loss of Cx43 staining appeared to be most stronglycounteracted when Hrs and Tsg101 were simultaneously depleted(Fig. 4B). Similarly to what was observed when Hrs or Tsg101 weresingly depleted, under these conditions Cx43 was often found tolocalize in EEA1-positive endosomes (Fig. 5).

In agreement with previous studies, depletion of Hrs and/orTsg101 resulted in larger endosomes (Fig. 5; and data not shown)(Razi and Futter, 2006). The degree of enlargement was greatestwhen both Hrs and Tsg101 were depleted. Importantly, under theseconditions most Cx43 was found to localize at the rim of theseenlarged endosomes (Fig. 5).

To gain a clearer understanding of the role of Hrs and Tsg101in degradation of Cx43 gap junctions, the ultrastructural localizationof Cx43 was determined by immunoelectron microscopy. In theseexperiments, control siRNA-transfected cells or cells depleted ofHrs and Tsg101 were treated with TPA and cycloheximide for 1.5hours. Cells were then prepared for electron microscopy, and Cx43was detected using immunogold particles. As expected, control cellswere nearly completely devoid of Cx43 labelling under theseconditions (data not shown). By contrast, cells depleted of Hrs andTsg101 showed strong subcellular Cx43 labelling. In accordancewith the confocal microscopy studies, Cx43 was frequently localizedin endosomes (Fig. 6A; supplementary material Fig. S2A). Inaddition, Cx43 was often found to be organized as annular gapjunctions (Fig. 6B; supplementary material Fig. S2B). Usually, thedouble membrane structure of these annular gap junctions appearedto be partly or completely disrupted, forming single membranes aswell as small intralumimal vesicles. Importantly, such annular gap

junctions often appeared to be in the process of fusing with othertypes of vesicles (Fig. 6C; supplementary material Fig. S2C). Othervesicles appeared to contain Cx43-enriched multivesicularstructures, presumably remnants of annular gap junctions (Fig. 6D;supplementary material Fig. S2D).

Interestingly, after prolonged treatment with TPA, most Cx43was found at the plasma membrane rather than in early endosomes(3 hours, Fig. 4B; 4.5 hours, supplementary material Fig. S3) incells depleted of Hrs or Tsg101. Under these conditions Cx43 wasorganized as distinct gap junctions. Also when Hrs and Tsg101 weresimultaneously depleted, a subpopulation of Cx43 was found to beorganized as gap junctions after 3 and 4.5 hours treatment of TPA(Fig. 4B and supplementary material Fig. S3, respectively).However, most Cx43 was localized in intracellular vesicles underthese conditions, in contrast to what was observed when Hrs andTsg101 were singly depleted.

Fig. 5. Depletion of Hrs and Tsg101 blocks trafficking of Cx43 from earlyendosomes to lysosomes. IAR20 cells were transfected with control siRNA orwith siRNA against Hrs or Tsg101 alone or simultaneously. At 48 hours aftertransfection, cells were co-incubated with TPA (100 ng/ml) and cycloheximide(chx, 10M) for 1.5 hours. Cells were double-stained with anti-Cx43antibodies and anti-EEA1 antibodies as indicated and visualized usingconfocal immunofluorescence microscopy. Merged images are shown in theright panels, with yellow indicating colocalization. Scale bars: 5m.

Fig. 6. Ultrastructural location of Cx43 as seen by immunoelectronmicroscopy. IAR20 cells were simultaneously transfected with siRNA againstHrs and Tsg101. After 48 hours of transfection, cells were co-incubated withTPA (100 ng/ml) and cycloheximide (10M) for 1.5 hours. Cells wereprepared for immunoelectron microscopy and Cx43 was detected on ultrathincryosections using 10 nm immunogold particles. (A)Cx43 labeling in anendosome. (B)Cx43 labeling in an annular gap junction. (C)Cx43 labeling inan annular gap junction that appears to have fused with a single-membranevesicle (arrowheads). (D)Cx43 labeling in what appears to be a partlydegraded annular gap junction within a larger endosome. Scale bars: 200 nm.

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PKC-induced degradation of Cx43 is dependent on Hrs andTsg101To substantiate the role of Hrs and Tsg101 in degradation of Cx43,we next examined how depletion of Hrs and/or Tsg101 affected theCx43 protein level, as determined by western blotting. Depletionof Tsg101 caused an approximate 50% increase in the total Cx43protein compared with control cells. By contrast, depletion of Hrsalone or in combination with Tsg101 did not significantly affectthe level of Cx43 protein (Fig. 7).

We next evaluated how depletion of Hrs and Tsg101 affected thePKC-induced degradation of Cx43. As expected, degradation ofCx43 in control siRNA-transfected cells was strongly induced inresponse to TPA treatment. After 1.5 hours chase, the Cx43 proteinlevel was approximately 25% that of the untreated cells (Fig. 7).Importantly, cells depleted of either Hrs or Tsg101 showed reduceddegradation of Cx43 in response to TPA exposure, and were foundto have a Cx43 protein level at this time point of approximately60% and 80%, respectively, compared to untreated control cells.The strongest effect on TPA-induced degradation of Cx43 wasobserved when Hrs and Tsg101 were simultaneously depleted, inwhich the Cx43 protein level was approximately 90% comparedwith untreated control cells.

After 3 hours of TPA treatment, the Cx43 protein level in controlsiRNA-transfected cells had decreased to approximately 10% ofthat in the untreated control cells (Fig. 7). At this time point, theCx43 protein level in cells depleted of Hrs or Tsg101 wasapproximately 40% and 55%, respectively, of that in the untreatedcontrol cells. Under conditions where Hrs and Tsg101 weresimultaneously depleted, the Cx43 protein level was approximately

80%. Collectively, these data strongly suggest that Hrs and Tsg101are both required for PKC-induced degradation of Cx43.

Interestingly, although both Hrs and Tsg101 depletioncounteracted the PKC-induced degradation of Cx43, the SDS-PAGEband pattern of Cx43 differed under these conditions. Following 3hours of TPA treatment, Cx43 was mainly found in the P0 and P1states in Hrs-depleted cells, whereas in Tsg101-depleted cells, mostCx43 was in the P1 and P2 states (Fig. 7). When both Hrs and Tsg101were depleted, Cx43 was predominantly found in the P2 state.

Hrs and Tsg101 regulate the level of gap junctionalintercellular communicationTo further elucidate the role of Hrs and Tsg101 in the endocytictrafficking of Cx43, we examined how depletion of Hrs and Tsg101affected the detergent resistance of Cx43. As expected, in untreatedcells, most P1 and P2 forms of Cx43 were Triton X-100 insoluble(Fig. 8A). After 1.5 hours of treatment with TPA and cycloheximide,Cx43 was mostly found in a Triton X-100 soluble form, inaccordance with the above data showing that at this time point Cx43is mostly localized in intracellular vesicles (Figs 4 and 5). Asexpected, the TPA-induced solubility of Cx43 in Triton X-100 wasnot counteracted by Hrs and/or Tsg101 depletion. Interestingly, after3 hours of treatment with TPA and cycloheximide, most Cx43 wasagain in a Triton X-100 resistant form in cells depleted of eitherHrs or Tsg101. This observation is in accordance with theimmunofluorescence data, showing that most Cx43 at this time pointis reorganized as gap junctions at the plasma membrane (Fig. 4B).By contrast, when Hrs and Tsg101 were simultaneously depleted,a significant subpopulation of Cx43 remained in a Triton X-100soluble state at this time point, in agreement with the data showingthat under these conditions Cx43 is localized both intracellularlyand in gap junctions (Fig. 4).

We next investigated whether the gap junctions present duringprolonged TPA treatment in Hrs- or Tsg101-depleted cells arefunctional. Depletion of Hrs and Tsg101 alone or in combinationdid not have major effects on gap junctional communication underconstituitive conditions (Fig. 8B). In accordance with previousstudies, TPA nearly completely blocked gap junctionalcommunication (supplementary material Fig. S4) (Rivedal andOpsahl, 2001). As expected, depletion of Hrs or Tsg101 did notaffect the initial TPA-induced block in communication. Following3 hours of exposure to TPA and cycloheximide, the gap junctionalcommunication in control siRNA-transfected cells remainedstrongly downregulated (Fig. 8C). Importantly, cells depleted of Hrsor Tsg101 showed an approximate threefold increase in gapjunctional communication compared with control siRNA-transfected cells at this time point. Taken together, these data suggestthat the gap junctions present during prolonged TPA treatment incells depleted of Hrs or Tsg101 are functional.

Deubiquitylation of Cx43 requires either Hrs or Tsg101Cx43 ubiquitylation in response to TPA treatment has previouslybeen shown to be transient (Leithe and Rivedal, 2004b). Themechanisms involved in the deubiquitylation of Cx43 are, however,not known. Since the above findings suggest an important role ofHrs and Tsg101 in the endocytic trafficking of Cx43, we askedwhether these proteins could play a role in regulating thedeubiquitylation of Cx43. Interestingly, under conditions where Hrsand Tsg101 were simultaneously depleted, Cx43 remained stronglyubiquitylated compared with control cells or cells singly depletedof Hrs or Tsg101 (Fig. 9A,B). This effect was observed both in


Fig. 7. PKC-induced degradation of Cx43 is dependent on Hrs and Tsg101.IAR20 cells were transfected with control siRNA or with siRNA against Hrs orTsg101 alone or simultaneously. After 48 hours of transfection, cells were leftuntreated or treated with TPA (100 ng/ml) and cycloheximide (chx, 10M) for1.5 or 3 hours. Cell lysates were prepared, and equal amounts of total cellprotein were subjected to SDS-PAGE. Cx43 was detected by western blotting,using Cx43 antibodies. The Cx43-P0, -P1 and -P2 bands are indicated. Theintensities of the Cx43 bands were measured using Scion Image. Values shownare the mean ± s.d. of three independent experiments. Statistical analysis wasperformed using one-way analysis of variance (ANOVA) with the Bonferronimultiple comparisons test. The asterisks indicate values that are significantlydifferent from those of control siRNA-transfected cells at the respective timepoint. (*P


untreated cells and cells treated with TPA for 1.5 or 3 hours.Importantly, Cx43 was found, under these conditions, to partlycolocalize with ubiquitin at the rim of enlarged endosomes, asdetermined by confocal microscopy (Fig. 9C). These data suggestthat either Hrs or Tsg101 must be present for Cx43 to undergodeubiquitylation. The data further suggest that under conditionswhere both Hrs and Tsg101 are absent, a subpopulation ofubiquitylated Cx43 accumulates at the rim of early endosomes.

Proteasomal activity is required for recruitment of ubiquitin togap junction plaques, but not for trafficking of Cx43 from earlyendosomes to lysosomesPrevious studies indicate an important role of the proteasome intrafficking of growth factor receptors from early endosomes to

lysosomes (Bishop et al., 2002; Longva et al., 2002; Rocca et al.,2001). Since the above data suggest that early endosomes have acrucial role in trafficking of ubiquitylated Cx43 to lysosomes, weconsidered it important to elucidate the role of the proteasome inthis process. We first investigated how proteasomal inhibitorsaffected the colocalization of ubiquitin and Cx43 gap junctionplaques, since our previous data indicated that proteasomal inhibitorscounteract TPA-induced ubiquitylation of Cx43 (Leithe and Rivedal,2004b). Interestingly, the proteasomal inhibitor MG132 stronglycounteracted ubiquitin recruitment to gap junctions, as determinedby confocal microscopy (Fig. 10A). Proteasomal inhibitors havebeen reported to reduce the cellular level of free ubiquitin, becauseof prevention of deubiquitylation of proteasomal substrates(Schubert et al., 2000). In accordance with these observations,treating IAR20 cells with TPA in combination with MG132 for 15minutes resulted in a strong reduction in the level of free ubiquitin(Fig. 10B). This observation suggests that the reduced recruitmentof ubiquitin to gap junction plaques caused by proteasomalinhibition is due to depletion of the pool of free ubiquitin.

To investigate whether the proteasome is involved in traffickingof Cx43 from early endosomes to lysosomes, cells were treatedwith TPA for 30 minutes and subsequently incubated for 60 minuteswith TPA alone or in combination with MG132 (Fig. 10B). Asexpected, in cells treated with only TPA for 90 minutes, most Cx43staining was lost. Interestingly, this loss of Cx43 was not affectedwhen TPA was co-incubated with MG132 for 60 minutes followingthe initial TPA treatment of 30 minutes. Collectively, these resultssuggest that once Cx43 has been ubiquitylated at the plasmamembrane, proteasomal activity is not required for its traffickingfrom the early endosome to the lysosome.

DiscussionModulation of gap junction degradation is considered to be animportant mechanism by which the level of intercellularcommunication via gap junctions is regulated (Berthoud et al., 2004;Laird, 2005; Musil et al., 2000; Thomas et al., 2003). However, themolecular machinery involved in the degradation of gap junctionshas remained poorly understood. It was previously reported thatEGF- and TPA-induced degradation of Cx43 is preceded by Cx43ubiquitylation (Leithe and Rivedal, 2004a; Leithe and Rivedal,2004b). In the present work, we have identified the ubiquitin-bindingproteins Hrs and Tsg101 as crucial mediators of Cx43 traffickingto the lysosome. To the best of our knowledge, this study representsthe first evidence that the early endosome functions as a sortingorganelle for ubiquitylated Cx43. The data provide new insight intohow cell-cell communication via gap junctions can be regulated atthe level of connexin turnover.

Activation of PKC was found to be associated with a dramaticrelocalization of ubiquitin to gap junction plaques, as determined byconfocal microscopy. Ubiquitin recruitment was particularlyprominent in gap junction regions that were in the process ofinternalizing into one of the adjacent cells. Often, entire gap junctionplaques were coated with ubiquitin in response to PKC activation.Furthermore, internalization of gap junctions was found to occur notonly in the centre of the plaque, but also in its periphery. Notably,this finding is in contrast to a previous study suggesting that only thecentre of the plaque is internalized, whereas the more peripheral partsof the plaque do not undergo internalization (Gaietta et al., 2002).The discrepancies between the two studies may possibly reflect twodifferent pathways for internalization of Cx43 gap junctions. The firstpathway, in which only Cx43 in the centre of the plaque is internalized,

Fig. 8. Re-assembly of Cx43 into functional gap junctions in Hrs- and Tsg101-depleted cells. (A)IAR20 cells were transfected with control siRNA or withsiRNA against Hrs or Tsg101 alone or simultaneously. After 48 hours oftransfection, cells were left untreated or treated with TPA (100 ng/ml) andcycloheximide (chx, 10M) for 1.5 or 3 hours. Cells were subsequentlysubjected to a Triton X-100 solubility assay. The Triton-X-100-soluble (S)and -insoluble (I) fractions were subjected to SDS-PAGE. Cx43 was detected bywestern blotting, using anti-Cx43 antibodies. (B,C)IAR20 cells were transfectedwith siRNA against Hrs or Tsg101, or with control siRNA. After 48 hours oftransfection, cells were (B) left untreated or (C) treated with TPA (100 ng/ml)and cycloheximide (chx, 10M) for 3 hours, and the level of gap junctioncommunication was determined. Values shown are the mean ± s.e.m. of threeindependent experiments. Statistical analysis was performed using one-wayanalysis of variance (ANOVA) with the Bonferroni multiple comparisons test.The asterisks indicate values that are significantly different from those of controlsiRNA-transfected cells at the respective time point. (*P

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might occur under constitutive conditions, possibly in a ubiquitin-independent manner. The second pathway, as reported here, mightpossibly be induced only in response to Cx43 phosphorylation andubiquitylation. The finding that ubiquitin can be present in gapjunction plaques is in agreement with a previous immunoelectronmicroscopy study by Rütz and Hülser (Rütz and Hülser, 2001).

Cx43 was found to remain ubiquitylated during its trafficking toearly endosomes, and partly colocalized with Hrs and Tsg101.Depletion of Tsg101 caused increased levels of Cx43 protein underconstitutive conditions. Interestingly, under these conditions Cx43was not found to localize in endosomal compartments, but wasinstead organized as intercellular gap junctions. Furthermore,although depletion of Tsg101 counteracted TPA-induced traffickingof Cx43 from the early endosome, Cx43 appeared to be able toescape early endosomes and relocate to the plasma membrane duringprolonged TPA treatment. Importantly, the localization of Cx43 atthe plasma membrane during prolonged treatment with TPA wasassociated with a normalization of the Cx43 phosphorylation and

ubiquitylation status. Furthermore, both the analysis of the detergentresistance of Cx43 and the dye transfer experiments indicate thatthe Cx43 population that escapes degradation under conditionswhere Tsg101 is depleted is able to form functional gap junctions.Further studies are required to elucidate the molecular mechanismsinvolved in the ability of Cx43 to form functional gap junctionsduring prolonged TPA treatment of cells depleted of Tsg101.However, one possibility could be that Cx43 undergoes recyclingfrom early endosomes to the plasma membrane. A scenario in whichCx43 undergoes trafficking from the early endosome to the plasmamembrane in cells lacking Tsg101 would be in accordance withwhat has been observed for other transmembrane proteins in othercell types. For instance, endocytosed EGF receptors that werenormally sorted to the lysosome were instead rapidly recycled backto the cell surface in a Tsg101 mutant cell line (Babst et al., 2000).Enhanced recycling of EGF receptor was also observed whenTsg101 was depleted by RNA interference (Bishop et al., 2002;Raiborg et al., 2008). The most common method used for studying


Fig. 9. Simultaneous depletion of Hrs and Tsg101 causesaccumulation of ubiquitylated Cx43. (A)IAR20 cells weretransfected with control siRNA or with siRNA against Hrs orTsg101 alone or simultaneously. After 48 hours of transfection,cells were left untreated or treated with TPA (100 ng/ml) andcycloheximide (chx, 10M) for 1.5 or 3 hours. Cells were thensubjected to immunoprecipitation with anti-Cx43 antibodies.Equal amounts of immunoprecipitates were subjected to SDS-PAGE, and ubiquitin was detected by western blotting using P4D1anti-ubiquitin antibodies (upper panel). The blot was stripped andreprobed with anti-Cx43 antibodies (lower panel).(B)Quantification of ubiquitylated Cx43 based on theimmunoprecipitation study in A. IAR20 cells were transfectedwith control siRNA or with siRNA against Hrs or Tsg101 alone orsimultaneously and treated with TPA and cycloheximide asdescribed in A. For each treatment, the level of ubiquitinimmunoreactivity was normalized to the level of Cx43immunoreactivity. Values shown are the mean ± s.d. of threeindependent experiments. Statistical analysis was performed usingone-way analysis of variance (ANOVA) with the Bonferronimultiple comparisons test. Cells simultaneously depleted of Hrsand Tsg101 have significantly higher levels of ubiquitylated Cx43protein than control siRNA-transfected cells (*P


recycling of plasma membrane proteins is to covalently link biotinmoieties to their extracellular domains followed by pulse-chaseexperiments. Biotinylation assays have been successfully used toshow that Cx43 hemichannels undergo recycling under specificconditions (VanSlyke and Musil, 2005). However, the cellularcompartment(s) in which recycling of Cx43 hemichannels occurshave not been identified. Unfortunately, biotinylation assays cannotbe used to investigate endocytosis and recycling of gap junction-associated Cx43, since the assembly of connexin proteins into gapjunction plaques prevents the binding of biotin to their extracellulardomains. Thus, although the data in the present study opens thepossibility that Cx43 can undergo recycling from the earlyendosomes to the plasma membrane, further studies are requiredto test this hypothesis.

While the present work was in progress, it was reported thatTsg101 binds to Cx31, Cx43 and Cx45 (Auth et al., 2009). It was

also suggested that Tsg101 is involved in constitutive degradationof Cx43 and Cx45, but not Cx31. Our results are in accordancewith the notion that Tsg101 plays a key role in Cx43 degradationunder constitutive conditions.

In contrast to Tsg101, Hrs depletion did not counteract theconstitutive degradation of Cx43. However, similarly to what wasobserved when Tsg101 was depleted, TPA-induced degradation ofCx43 was reduced in Hrs-depleted cells. Also under theseconditions, most Cx43 was found to localize at the plasmamembrane during prolonged TPA treatment. The TPA-induceddegradation of Cx43 was most strongly counteracted when Hrs andTsg101 were simultaneously depleted. Interestingly, under theseconditions, most Cx43 appeared to remain localized in earlyendosomes in a phosphorylated and ubiquitylated state, even afterprolonged TPA treatment. It should be noted that thephosphorylation events involved in the formation of the Cx43-P2form are currently incompletely understood (Solan and Lampe,2009). Thus, the Cx43 phosphorylation sites that are responsiblefor the formation of the Cx43-P2 band observed during prolongedTPA treatment of cells depleted of Hrs and Tsg101 remain to bedetermined. Further studies are also required to elucidate therelationship between Cx43 phosphorylation and ubiquitylation, bothunder constitutive conditions and in response to TPA treatment.

Taken together, the present data identify the early endosome asa major sorting station for ubiquitylated Cx43 and reveal a crucialrole of Hrs and Tsg101 in trafficking of Cx43 to lysosomes. It is

Fig. 10. Proteasomal inhibition counteracts recruitment of ubiquitin to Cx43gap junction plaques. (A)IAR20 cells were treated with TPA (100 ng/ml)alone or co-incubated with TPA and MG132 (10M) for 15 minutes. Cellswere double-stained with anti-Cx43 antibodies and FK2 anti-ubiquitinantibodies and visualized using confocal immunofluorescence microscopy.Merged images are shown in the right panels, with yellow indicatingcolocalization. Scale bars: 5m. (B)IAR20 cells were treated with TPA (100ng/ml) alone or co-incubated with TPA and MG132 (10M) for 15 minutes asindicated. Cell lysates were prepared, and equal amounts of total cell proteinwere subjected to SDS-PAGE. Ubiquitin was detected by western blottingusing P4D1 antibodies and actin was detected with anti-actin antibodies.(C)IAR20 cells were left untreated, treated with TPA (100 ng/ml) for 30minutes, or treated with TPA for 30 minutes and subsequently treated withTPA and DMSO (vehicle) or MG132 (10M) for 60 minutes, as indicated.Cells were stained with anti-Cx43 antibodies and visualized usingimmunofluorescence microscopy.

Fig. 11. Proposed model for the role of ubiquitin in Cx43 degradation. Gap-junction-associated Cx43 is covalently conjugated to ubiquitin at the plasmamembrane. Cx43 remains ubiquitylated as gap junctions internalize into one ofthe adjacent cells and during the maturation of the annular gap junction(connexosome) double-membrane structure into single membranes. Wepropose that trafficking of ubiquitylated Cx43 to late endosomes andlysosomes is mediated by Hrs and Tsg101. We also propose that in the absenceof Hrs or Tsg101, Cx43 can undergo dephosphorylation and deubiquitylationat early endosomes, and participate in the formation of functional gapjunctions, possibly by recycling of Cx43 from early endosomes.

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thought that Hrs and Tsg101 mediate trafficking of growth factorreceptors by binding directly to ubiquitylated receptors in the earlyendosome (Raiborg and Stenmark, 2009). In analogy to their rolein downregulation of growth factor receptors, we propose a modelin which Hrs and Tsg101 mediate lysosomal degradation of Cx43by binding to ubiquitylated Cx43 (Fig. 11). This study furthersuggests that either Hrs or Tsg101 must be present for Cx43 toundergo deubiquitylation, possibly by recruiting enzymes catalyzingCx43 deubiquitylation.

The human genome encodes 84 predicted active deubiquitylationenzymes, several of which have been shown to localize at earlyendosomes and mediate deubiquitylation of internalized receptors(Saksena et al., 2007). An important subject for future research willbe to identify the enzymes catalyzing Cx43 deubiquitylation, andtheir potential role in regulating the level of gap junctionalcommunication.

Materials and MethodsCell cultureThe rat liver epithelial cell line IAR20, originally isolated from normal inbred BD-IV rats (Asamoto et al., 1991; Montesano et al., 1977), was obtained from theInternational Agency for Research on Cancer (Lyon, France). Cells were grown inDulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetalbovine serum (FBS; Gibco BRL Life Technologies, Inchinnan, UK). The growthmedium was replaced with DMEM supplemented with 1% (v/v) FBS 24 hours priorto experiments.

siRNAThe siRNA oligonucleotides targeted against Hrs or Tsg101 were obtained fromInvitrogen (Stealth Select RNAi HGSRSS329127 and TSG101RSS333993,respectively). The Hrs siRNA had the following sequence: 5�-UUAUCAUUG ACC -UUCUUCUUGAUGG-3�. The sequence of the Tsg101 siRNA was: 5�-CCA -GGCAGAGCUUAAUGCCUUGAAA-3�. The siRNA control construct was StealthRNAi Negative Control Medium GC (Invitrogen). siRNA was transfected into cellsusing Lipofectamine 2000 (Invitrogen), according to the manufacturer’s direction, ata final concentration of 80 nM. In some experiments, siRNA against Hrs and Tsg101were co-transfected into cells, at a total final concentration of 80 nM. IAR20 cellsgrown in 35 mm Petri dishes were transfected 24 hours after seeding. The growthmedium was replaced with DMEM supplemented with 1% (v/v) FBS 24 hours aftertransfection. The cells were assayed 48 hours after transfection. To ensure that theeffect of Hrs and Tsg101 depletion on Cx43 as reported in the present study was nota result of an off-target effect, we repeated the experiments with two independentsiRNA duplexes. In these experiments, we obtained a similar effect on Hrs and Tsg101depletion and on Cx43 trafficking as with the siRNA duplexes described above,indicating that the observed effect of siRNA on Cx43 was indeed due to depletionof Hrs and Tsg101 (data not shown).

Reagents and antibodiesTPA, cycloheximide, Lucifer yellow and chlordane were obtained from Sigma (StLouis, MO). GF109203 was from Calbiochem (La Jolla, CA). Anti-Cx43 antibodieswere obtained by injecting rabbits with a synthetic peptide consisting of the 20 C-terminal amino acids of Cx43 (Rivedal et al., 1996). Mouse anti-Cx43 antibodieswere from Chemicon International (Temecula, CA). The P4D1 (mouse IgG) and FK2(mouse IgG) anti-ubiquitin antibodies were obtained from Covance (Berkeley, CA)and Affinity Research Products (Exeter, UK), respectively. Mouse anti-earlyendosomal autoantigen-1 (EEA1) antibodies were from BD transduction laboratories(San Diego, CA). Rabbit anti-Hrs antibodies have been described previously (Raiborget al., 2001). Mouse anti-Tsg101 antibodies were purchased from Genetex (SanAntonio, TX) or Abcam (4A10, Cambridge, UK). Mouse anti-actin antibodies werefrom Sigma. Alexa-Fluor-488-conjugated goat anti-rabbit IgG and Alexa-Fluor-594-conjugated goat anti-mouse IgG were from Molecular probes (Eugene, OR).Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies werefrom Bio-Rad (Hercules, CA). Horseradish peroxidase-conjugated donkey anti-mouseIgG antibodies were obtained from Jackson Immunoresearch Laboratories (WestGrove, PA).

Western blotting and immunoprecipitationWestern blotting and immunoprecipitation was performed as described previously(Leithe and Rivedal, 2004a). The intensities of Cx43 bands were quantified usingScion Image (Scion). In some experiments, cells were subjected to a Triton X-100solubility assay, as described below, before immunoprecipitation.

Analysis of the Cx43 detergent resistanceThe Triton X-100 solubility of Cx43 was determined based on a method describedby VanSlyke and Musil (VanSlyke and Musil, 2000), as described previously (Sirneset al., 2008). Briefly, cells were collected in 1 ml incubation buffer (136.8 mM NaCl,5.4 mM KCl, 0.34 mM Na2HPO4, 0.35 mM KH2PO4, 0.8 mM MgSO4, 2.7 mMCaCl2, 20 mM HEPES, pH 7.4) containing 10 mM N-ethylmaleimide and 200 Mphenylmethylsulfonyl fluoride and centrifuged at 3000 g for 4 minutes at 4°C. Thecells were resuspended in 400 l incubation buffer containing 10 mM N-ethylmaleimide, 200 M phenylmethylsulfonyl fluoride, 22 M leupeptin andphosphatase cocktail, and sonicated for 10 seconds. Triton X-100 was added to afinal concentration of 1% (v/v). Lysates were incubated for 30 minutes at 4°C. A200 l aliquot of the sample was then transferred to a microcentrifuge tube andcentrifuged at 100,000 g for 50 minutes at 4°C. The supernatant fraction was collectedand the pellet was resuspended in 200 l incubation buffer containing 10 mM N-ethylmaleimide and 200 M phenylmethylsulfonyl fluoride. To all three fractions(total cell lysate, Triton-X-100-soluble and Triton-X-100-insoluble), 200 l samplebuffer containing 2� SDS was added and heated for 5 minutes at 95°C. Western blotanalysis was performed as described above.

Immunofluorescence and confocal microscopyCells were fixed and stained as described previously (Leithe and Rivedal, 2004a).Nuclei were stained with Hoechst 33342. Immunofluorescence images were capturedusing a Nikon E800 microscope with a Spot-2 camera. For colocalization studies,cells fixed on coverslips were analyzed with a LSM 510 META confocal microscope(Carl Zeiss) equipped with Plan Apochromat 63� 1.4 NA and Neo Fluar 100� 1.45NA oil immersion objectives (Carl Zeiss). Appropriate emission filter settings wereincluded to exclude bleed-through effects. Images were acquired with the LSM 510software (version 3.2; Carl Zeiss) and processed with Adobe Photoshop, version 7.0.

Immunoelectron microscopyCells were prepared for immunoelectron microscopy as described previously (Leitheet al., 2006a). Cryosections were transferred to formvar-carbon-coated grids andlabelled with anti-Cx43 antibodies followed by protein A-gold conjugates. The labelledcryosections were examined with a Philips CM10 or a JEM 1230 (JEOL) electronmicroscope.

Determination of gap junctional communication by quantitativescrape loadingQuantitative scrape loading was performed as previously described (Leithe et al.,2003; Opsahl and Rivedal, 2000). Digital monochrome images were acquired usinga COHU 4912 CCD camera (COHU, San Diego, CA) and a Scion LG-3 frame grabbercard (Scion Corporation, Frederick, MD).

We are grateful to Astri Nordahl, Zeremariam Yohannes andMarianne Smestad for excellent technical assistance, to Camilla Raiborg(Norwegian Radium Hospital, Oslo, Norway) for kindly providing theanti-Hrs antibody, and to Sharmini Alagaratnam for critical reading ofthe manuscript. This work was supported by the Norwegian CancerSociety and the Research Council of Norway.

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Ubiquitylation of the gap junction protein connexin-43 signals its … · 2009. 11. 4. · (Fig. 1). The two upper bands (Cx43-P1 and Cx43-P2) represent Cx43 organized in gap junctions