Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its lysosomal degradation

Abstract

Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts. Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts. Normal organ function depends on intercellular communication facilitated by channels contained within gap junctions that allow direct intercellular diffusion of ions and small molecules up to ∼1 kDa (including many intracellular signaling molecules). These channels are formed by members of a family of related integral membrane proteins called connexins. The connexins contain four transmembrane domains with the amino and carboxyl termini localized in the cytoplasm. The biological importance of connexins and the intercellular communication that they support is emphasized by the linkage of mutations in different connexin genes to diverse human diseases, including oculodentodigital dysplasia, deafness, cataracts and diseases of skin, nervous system, and cardiovascular system (reviewed in Refs. (1Delmar M. Makita N. Cardiac connexins, mutations and arrhythmias.Curr. Opin. Cardiol. 2012; 27: 236-241Google Scholar, 2Srinivas M. Verselis V.K. White T.W. Human diseases associated with connexin mutations.Biochim. Biophys. Acta Biomembr. 2018; 1860: 192-201Google Scholar, 3Ehrlich A. Molica F. Hautefort A. Kwak B.R. Lymphatic connexins and pannexins in health and disease.Int. J. Mol. Sci. 2021; 22: 5734Google Scholar)). Studies of wild-type and mutant connexins expressed in cells or in animal models have provided a significant body of information regarding their function, life cycle, and regulation, and the contributions of the different connexin polypeptide domains to these processes. To elucidate how alterations of connexins contribute to disease, we have been studying two connexins that are almost uniquely expressed in the lens, connexin46 (Cx46) and connexin50 (Cx50). The lens is formed by an anterior epithelial cell layer and fiber cells that comprise the bulk of the organ. The normal healthy lens is a transparent avascular organ that relies on gap junction–mediated intercellular communication to maintain homeostasis and transparency by supporting the circulation of water and ions within the organ (reviewed in Refs. (4Mathias R.T. Kistler J. Donaldson P. The lens circulation.J. Membr. Biol. 2007; 216: 1-16Google Scholar, 5Mathias R.T. White T.W. Gong X. Lens gap junctions in growth, differentiation, and homeostasis.Physiol. Rev. 2010; 90: 179-206Google Scholar, 6Berthoud V.M. Gao J. Minogue P.J. Jara O. Mathias R.T. Beyer E.C. Connexin mutants compromise the lens circulation and cause cataracts through biomineralization.Int. J. Mol. Sci. 2020; 21: 5822Google Scholar)). A large number of Cx46 and Cx50 mutants have been identified in families of people with inherited congenital cataracts. Like the mutants of other connexins associated with disease, many of the cataract-linked Cx46 and Cx50 mutants do not traffic properly and are degraded or accumulate in intracellular compartments; therefore, they are unable to support intercellular communication. Some mutants traffic to the plasma membrane properly and form gap junctional plaques, but they are not functional or have altered gap junctional channel or hemichannel properties (reviewed in Ref. (7Berthoud V.M. Ngezahayo A. Focus on lens connexins.BMC Cell Biol. 2017; 18: 6Google Scholar)). Many of the cataract-linked mutations that have been previously studied are missense substitutions of amino acids in the first half of the molecule (from the amino terminus through the fourth transmembrane domain). These studies have helped to elucidate specific structure–function relationships in the connexins. Only a small number of cataract-linked mutations have been identified in the carboxyl terminal domain, even though this domain corresponds to a large fraction of the connexin protein (∼50% for Cx50), and the mechanisms by which they lead to disease have only been studied for a few of them (8Minogue P.J. Liu X. Ebihara L. Beyer E.C. Berthoud V.M. An aberrant sequence in a connexin46 mutant underlies congenital cataracts.J. Biol. Chem. 2005; 280: 40788-40795Google Scholar, 9Minogue P.J. Beyer E.C. Berthoud V.M. A connexin50 mutant, CX50fs, that causes cataracts is unstable, but is rescued by a proteasomal inhibitor.J. Biol. Chem. 2013; 288: 20427-20434Google Scholar, 10Liu Y. Qiao C. Wei T. Zheng F. Guo S. Chen Q. Yan M. Zhou X. Mutant connexin 50 (S276F) inhibits channel and hemichannel functions inducing cataract.J. Genet. 2015; 94: 221-229Google Scholar). The carboxyl terminus of all connexins is located within the cytoplasm and has been implicated in various protein–protein interactions (reviewed in Refs. (11Solan J.L. Lampe P.D. Connexin43 phosphorylation: Structural changes and biological effects.Biochem. J. 2009; 419: 261-272Google Scholar, 12Hervé J.C. Derangeon M. Sarrouilhe D. Giepmans B.N. Bourmeyster N. Gap junctional channels are of Biophys. 2012; Scholar)). many sites for which phosphorylation is The and protein–protein interactions of the connexin life and channel (reviewed in Refs. (11Solan J.L. Lampe P.D. Connexin43 phosphorylation: Structural changes and biological effects.Biochem. J. 2009; 419: 261-272Google Scholar, 12Hervé J.C. Derangeon M. Sarrouilhe D. Giepmans B.N. Bourmeyster N. Gap junctional channels are of Biophys. 2012; Scholar)). In the we the and functional properties of two cataract-linked Cx50 Cx50S258F and Cx50S259Y L. A. P. T. in a of families with congenital Sci. 2009; Scholar, X. J. C. X. F. Liu C. M. S. X. A in the connexin 50 associated with congenital in a 2010; Scholar, J. Chen M. and of with congenital in stably transfected HeLa cells and These mutations serines in the carboxyl terminus of the protein studies of and have that the serines to and of human Cx50 are D. in lens fiber connexins identified by Sci. Scholar, and sites of lens connexin and connexin 2009; Scholar, J. and of human lens fiber cell Sci. 2013; Scholar, of lens connexin intercellular channels by 2018; Scholar). these mutants provided an to the function of phosphorylation at these positions and how to results that these mutants cause disease by Cx50 function through of a sorting signal that them to the a that has not previously been for connexin To the behavior of Cx50S258F and we stably transfected HeLa cells these mutants and These serines are in and D. in lens fiber connexins identified by Sci. Scholar, and sites of lens connexin and connexin 2009; Scholar, J. and of human lens fiber cell Sci. 2013; Scholar, of lens connexin intercellular channels by 2018; Scholar). to the of a that phosphorylation at these positions, we stably transfected HeLa cells Cx50 in which or by and To to the plasma membrane and of gap junctional plaques, we localized Cx50 by HeLa cells wild-type Cx50 at gap junctional plaques and in the In contrast, and cells Cx50 in the very they small plaques at membrane that were The number of membrane gap junctional plaques in but significantly in cells and cells In contrast, cells the aspartate substitution mutants and of gap junctional plaques The of cell gap junctional plaques in cells and in cells in cells the of gap junctional plaques in the cells the aspartate substitution mutants and Cx50S259D) the in HeLa cells wild-type Cx50 The of the cataract-linked mutants that they localized in the To we with and in HeLa cells wild-type Cx50 or the Cx50 mutants. Some wild-type Cx50 with the but Cx50S258F and Cx50S259Y a to the protein of the cells with A that the the in of Cx50 as A treatment of and Cx50 the cell as the cells of intracellular and a few Cx50 gap junctional plaques We Cx50 levels in the HeLa by of Cx50 were significantly lower in and cells compared with in cells and Cx50, A and In contrast, levels of Cx50 in and cells were not significantly different cells wild-type Cx50 and the in these levels in the HeLa cells the cataract-linked mutants. In protein levels of Cx50 were the levels in in they were the levels in A and To these changes in levels, we Cx50 levels were the HeLa cells wild-type Cx50 and cells the cataract-linked mutant, Cx50S258F Cx50 levels were significantly lower in cells compared with cells but the than that of Cx50 protein levels for the protein The increased Cx50 levels in HeLa cells the aspartate substitution mutants not changes in Cx50 levels were and cells and and cells and These results that the Cx50 levels in cells the cataract-linked Cx50 mutants degradation of the the and the lysosome have been implicated in degradation of wild-type and mutant connexins (reviewed in Refs. V.M. Minogue P.J. Beyer E.C. for degradation of connexins and gap Scholar, Berthoud V.M. of connexins and gap Scholar)). To these were in the degradation of the Cx50 mutants, we HeLa cell with chloroquine that inhibits the of lysosomal by the of the or of and cells with for not lead to a significant in levels of Cx50 in of the HeLa cell to the Cx50S259Y levels were of the by which increased abundance of proteins treatment a large of Cx50 levels in the cells the mutants for Cx50S258F and for Cx50S259Y the levels in treatment to in cells wild-type Cx50 Cx50S258D and Cx50S259D A and treatment with an in Cx50 levels in all The in cells the cataract-linked mutants and levels of Cx50 increased in cells and in they increased in in and in cells treatment with for or to aspartate Cx50 protein levels to wild-type in A and and the of the protein that for lysosomal degradation by at the plasma We the of lysosomal on the of Cx50 in the HeLa Cx50 in a within the cytoplasm in cells wild-type Cx50 or of the Cx50 mutants treatment with chloroquine or with their lysosomal degradation. Some at detected in cells wild-type Cx50, or with the of gap junction plaques that were not To the of of and on Cx50 function, we gap junctional in of wild-type or mutant wild-type Cx50 large junctional compared with only with to the In contrast, Cx50S259Y not junctional detected in the and Cx50S258F junctional that were only of the by wild-type Cx50 for for wild-type the aspartate substitution mutants, Cx50S258D and junctional that were significantly than by wild-type Cx50 for and for for wild-type To intercellular transfer of small molecules in the Cx50 mutants, we studied intercellular transfer of in the stably transfected HeLa cells. Cx50S258F and Cx50S259Y supported transfer to a significantly number of cells than HeLa but to a significantly lower number of cells than wild-type Cx50 and cells in cells and cells cells in HeLa cells and cells in mutants intercellular transfer of the number of cells cells in or cells in than that cells wild-type Cx50 of a at amino positions or that phosphorylation of the protein to gap junctional plaques and function the protein to a for lysosomal we the that the terminus of Cx50 contained a signal that sorting signal by the cataract-linked which these phosphorylation we the amino sequence of the terminus of Cx50 for sorting We identified that to the tyrosine-based sorting signal of the in which is is amino and is an amino with a or (reviewed in Ref. for sorting of transmembrane proteins to and Rev. and To the that of these sorting in the cataract-linked Cx50 mutants to lysosomal we mutants of Cx50S258F and Cx50S259Y in which amino acids to were we of HeLa cells transfected with these mutants by we observed the of gap junctional plaques at that a sorting sequence localized amino acids to of the sequence as a is an in this of sorting and by other amino we mutants to the sorting amino acids and in Cx50S258F and Cx50S259Y by or with and a first we of HeLa cells these and them for the of gap junctional plaques by gap junctional plaques were observed in cells and of by not gap junction abundance implicated but not in the increased lysosomal sorting of the mutant We and with and this of sorting signal an amino with a at we of HeLa cells these substitution mutants, we observed gap junctional plaques. these results that the signal for the behavior of the cataract-linked mutants. for we of stably transfected HeLa cells the and HeLa cells stably transfected with the and mutants of Cx50S258F or Cx50S259Y gap junctional plaques The of plasma membrane gap junctional plaques in cells transfected with the mutants than that in wild-type Cx50 HeLa cell for for for for cells for In the of gap junctional plaques in the cells these mutants were significantly compared with in cells the substitution for in and the of gap junctional plaques and the for wild-type Cx50 gap junctional plaques. the substitutions for and in Cx50S258F and the and the in wild-type Cx50 To gap junction function in the or substitution mutants, we intercellular transfer of in the HeLa cell these mutants transfer to at the number of cells in wild-type Cx50 HeLa cells wild-type Cx50 to an of cells to cells to cells to and cells to cells. In this we have that the cataract-linked mutants, Cx50S258F and support or intercellular communication of the of gap junctional plaques at of function results of a tyrosine-based sorting signal and degradation of the protein in the Only small of the mutant protein are in gap junctional plaques they support a low of intercellular communication. The levels of the mutant protein are to by and than the levels of wild-type Cx50, increased participation of the lysosome in the degradation of the cataract-linked mutant studies have that and in Cx50 are in D. in lens fiber connexins identified by Sci. Scholar, and sites of lens connexin and connexin 2009; Scholar, J. and of human lens fiber cell Sci. 2013; Scholar, of lens connexin intercellular channels by 2018; Scholar). is a that has been implicated in of the life of connexins, including targeting the protein to the plasma of gap junction of gap and protein degradation (reviewed in Ref. P.D. The of connexin phosphorylation on gap junctional J. Cell Biol. Scholar)). of for or a not the of Cx50S258F and Cx50S259Y on connexin levels, gap junctional and intercellular communication. Gap junctional plaques are and gap junctional is these positions are by serine or by the of a of at these positions to the protein to the plasma membrane to gap junctional the the in the aspartate substitution the the targeting the protein to the lysosome for degradation. results suggest that the absence of the phosphorylatable serine at positions and in the cataract-linked Cx50 mutants to of a sorting signal that the protein for lysosomal degradation with a in the levels of Cx50 and the abundance of gap junctional plaques, which contain intercellular We identified the amino sequence acids as this sorting to the tyrosine-based sorting signal of the that is by the protein (reviewed in Ref. for sorting of transmembrane proteins to and Rev. Scholar)). of for this of or to in Cx50S258F and Cx50S259Y significantly the number of gap junctional plaques and We previously increased Cx50 levels and intercellular communication in HeLa cells stably a Cx50 amino compared with HeLa cells wild-type P.J. Beyer E.C. Berthoud V.M. A connexin50 mutant, CX50fs, that causes cataracts is unstable, but is rescued by a proteasomal inhibitor.J. Biol. Chem. 2013; 288: 20427-20434Google Scholar). These changes in the Cx50 by the of the tyrosine-based sorting the is the first that a in a connexin has the of a sorting the of tyrosine-based sorting previously predicted the of connexins and mechanisms and 2013; Scholar). studies of mutants have the importance of acids and acids for gap junction and N. N. M. O. A tyrosine-based sorting signal is in and gap junction Cell Sci. Scholar, tyrosine-based sorting in the to gap junction Biol. 2013; Scholar). and plasma membrane have been implicated in the tyrosine-based of gap junctions tyrosine-based sorting in the to gap junction Biol. 2013; Scholar). the large body of that in many other proteins are by the (reviewed in Ref. for sorting of transmembrane proteins to and Rev. and the studies of is that are in of Cx50 with the sorting has been that amino acids the sorting signal amino the signal in and the signal in has been implicated in targeting proteins tyrosine-based sorting to the lysosome for sorting of transmembrane proteins to and Rev. Scholar). with the that and are in the lens D. in lens fiber connexins identified by Sci. Scholar, and sites of lens connexin and connexin 2009; Scholar, J. and of human lens fiber cell Sci. 2013; Scholar, of lens connexin intercellular channels by 2018; a for lysosomal targeting and degradation of the mutants. We that the carboxyl terminus of wild-type Cx50 serine at or a that the sorting signal or of the lysosomal In contrast, in the cataract-linked mutants, Cx50S258F and the absence of a serine at of these sites phosphorylation and exposes the sorting signal leading to lysosomal targeting of the connexin and degradation. is that lysosomal targeting at the mutant protein is at a of the mutant protein has a and rarely of the protein detected gap junction plaques, is that the cataract-linked Cx50 mutants to the membrane protein protein a sorting signal and in the lysosome with low levels at the plasma membrane M. of membrane in a at a in the Cell Biol. but membrane protein the Cx50 mutants are degraded in the In the lens, increased of the sorting signal of of or lead to targeting of Cx50 to the lysosome and degradation. lens fiber cells do not contain degradation of the mutant Cx50 in the of fiber only the lens epithelial and fiber cells contain of Cx50 greatly abundance and the functions The human cataracts associated with these mutants are or they to the of the that the function is intercellular the lens is on the lens circulation of water and which gap junctions a of Cx50 alterations in and of fiber cells in the human lens as in Cx50 changes have been to the absence of to cell T.W. of connexin50 in results in and cataracts.J. Cell Biol. Scholar, S. S. L. Y. White T.W. M. Connexin 50 and are in and of lens Sci. Scholar). decreases in fiber cell gap junctional have been in and Gong X. Mathias R.T. Gap junctional in connexin Scholar, V.M. Minogue P.J. Beyer E.C. decreases levels of fiber cell connexins and lens fiber cell Sci. 2013; Scholar, V.M. Gao J. Minogue P.J. Jara O. Mathias R.T. Beyer E.C. The mutant causes cataracts by Sci. Scholar). In people the Cx50S258F and Cx50S259Y the cataracts are inherited as L. A. P. T. in a of families with congenital Sci. 2009; Scholar, X. J. C. X. F. Liu C. M. S. X. A in the connexin 50 associated with congenital in a 2010; Scholar, J. Chen M. and of with congenital Scholar). Cx50 have of the normal levels of are transparent T.W. of connexin50 in results in and cataracts.J. Cell Biol. Scholar, P. X. Y. Gong X. of in to associated with of lens and lens fiber Scholar). The of the cataracts in the Cx50S258F or Cx50S259Y mutations that these mutants cause a in Cx50 levels and function by than through of the Cx50 mutants and wild-type wild-type Cx50 in of the mutants to the plasma these of the of the is that the mutant Cx50 in causes degradation of wild-type Cx50 leading to studies have the importance of phosphorylation sites that in with a sorting serine or is the sorting signal of Cx50 in gap junction plaques. amino than phosphorylation at these positions lead to of the sorting signal and degradation of the The of Cx50S258F and Cx50S259Y with cataracts L. A. P. T. in a of families with congenital Sci. 2009; Scholar, X. J. C. X. F. Liu C. M. S. X. A in the connexin 50 associated with congenital in a 2010; Scholar, J. Chen M. and of with congenital that serine phosphorylation at these sites and the sorting signal function in and have a importance in the by which these mutants lead to other proteins contain phosphorylation sites and sorting is that of their mutations lead to a Cx50 were by were in to the mutations the The different mutants were the of the wild-type human as the V.M. Minogue P.J. Guo J. X. Ebihara L. Beyer E.C. of function and degradation of a mutant J. Cell Biol. and the The were or as the and or or or or were at the of the of to that not HeLa cells were in with amino and at were transfected with wild-type or mutant Cx50 in the To stably transfected were by their to To we many for wild-type Cx50, and Cx50S259D by the a of the We of of of of and of were to the with normal the chloroquine or A and for were on they they were in in for and to previously the carboxyl terminus of Cx50 acids V.M. Minogue P.J. Guo J. X. Ebihara L. Beyer E.C. of function and degradation of a mutant J. Cell Biol. or by or as previously V.M. Minogue P.J. Guo J. X. Ebihara L. Beyer E.C. of function and degradation of a mutant J. Cell Biol. Scholar). were studied with a of in an with a were with a and were for all within an of the that for the the signal were at by two different not the The number of gap junctional plaques and the of gap junctional plaques in cells stably transfected with were to of 2012; Scholar). The for a gap junction different up to were to the number of are as the of the in that contained gap junctional plaques. A of up to gap junctional plaques were to gap junctional The results are in were and for were are as were at to on of were with in and and at for were in and To levels of Cx50, cell of protein were to of protein and transfer by the with the for or were with previously V.M. Minogue P.J. Guo J. X. Ebihara L. Beyer E.C. of function and degradation of a mutant J. Cell Biol. by as previously P.J. A. Ebihara L. Beyer E.C. Berthoud V.M. A mutant connexin50 with hemichannel function to cell Sci. 2009; Scholar, V.M. Minogue P.J. Beyer E.C. The cataract-linked mutant causes in Biol. Chem. Scholar). of the detected of the protein and immunoblotting with are in that the kDa and kDa detected in Cx50 is To that the were of at for and with by The were by a that The size to the for all in an The size to a in and the as previously P.J. Beyer E.C. Berthoud V.M. is for lens transparency in wild-type and connexin50 mutant Scholar). The results are in were were are as at were in and the the were a the on a a and a of or Cx50 These an that within a and they not as by the contained of in in of The for the to the levels of the different HeLa were are as the of the Cx50 levels in the mutant HeLa cell compared with in the cells. To gap junctional coupling, the were the The were with and were the in to the The of by the at were with connexin were and as previously L. of gap junctional proteins in Scholar). To the junctional cell were studied the properties of junctional Scholar). were and an as previously X. Ebihara L. of a Cx50 associated with the Sci. Scholar). and were a and a system cells of the were at and were to cell the cell at The junctional were with and of to The the were on previously with to that the observed were not by the of L. channels in the plasma membrane of J. Scholar). the were at were for their the or HeLa cells were on they cell within a with a to the Cx50 to this and for a the gap junction to to cells for cells were in for and with to allow of by as previously P.J. Beyer E.C. Berthoud V.M. A connexin50 mutant, CX50fs, that causes cataracts is unstable, but is rescued by a proteasomal inhibitor.J. Biol. Chem. 2013; 288: 20427-20434Google Scholar). The of intercellular transfer by the number of cells the The number of for cells the different to are as are expressed as were for or A of are contained within the and supporting supporting The that they have of with the of this P. J. C. and M. P. J. M. and M. P. J. L. and M. P. J. M. and T. M. P. J. L. C. and M. P. J. M. and M. L. and M. C. L. C. and M. supported by the of C. and M. and L. supported by the for M. supported by a the of The of this is the of the and not the of the of

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