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Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its
Peter J Minogue1, Jun-Jie Tong2, Kelly Wichmann1
1Department of Pediatrics, University of Chicago, Chicago, Illinois, USA.
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.
Insights
Connexin50 (Cx50) mutants linked to cataracts are degraded via lysosomes when phosphorylation sites are absent. Phosphorylation at these sites stabilizes Cx50, preventing degradation and maintaining gap junction function, crucial for preventing cataracts.
Area of Science:
- Cell biology
- Ophthalmology
- Molecular genetics
Background:
- Connexin50 (Cx50) is a key protein in lens gap junctions.
- Mutations in Cx50 are associated with human cataracts.
- Specific serine residues (S258, S259) in Cx50 are phosphorylation sites linked to cataract development.
Purpose of the Study:
- To investigate the role of phosphorylation at Cx50 serine residues (S258, S259) in protein stability and function.
- To examine the impact of cataract-linked Cx50 mutants on gap junction formation and intercellular communication.
- To elucidate the degradation pathway of Cx50 mutants lacking phosphorylation sites.
Main Methods:
- Stable transfection of HeLa cells with wild-type Cx50 and various Cx50 mutants (S258F, S259Y, S258D, S259D).
- Analysis of gap junction plaque formation using microscopy.
- Assessment of intercellular communication via neurobiotin transfer.
- Immunoblotting to quantify Cx50 protein levels.
- Treatment with lysosomal inhibitors (chloroquine, ammonium chloride).
- Site-directed mutagenesis of a predicted tyrosine-based sorting signal.
Main Results:
- Cx50 mutants lacking phosphorylatable serines (S258F, S259Y) showed reduced gap junction plaques and low intercellular coupling.
- Phosphorylation-mimicking Cx50 mutants (S258D, S259D) exhibited increased gap junction abundance, size, and extensive communication.
- Cx50S258F and Cx50S259Y protein levels were reduced, with increased degradation via lysosomes.
- Mutating a tyrosine-based sorting signal in Cx50S258F/S259Y enhanced gap junction formation and communication.
- Phosphorylation at S258/S259 appears to mask a lysosomal degradation signal.
Conclusions:
- Absence of phosphorylation at Cx50 S258/S259 exposes a sorting signal, targeting Cx50 for lysosomal degradation.
- Phosphorylation at these sites stabilizes Cx50 by concealing the degradation signal, promoting plasma membrane targeting and gap junction plaque formation.
- Cx50 degradation due to lack of phosphorylation in lens cells likely reduces Cx50 function, contributing to cataract formation.
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