The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulum

Mutant connexins have been linked to hereditary congenital cataracts. One such mutant causes a proline-to-serine substitution at position 88 in human connexin 50 (CX50P88S). In transfected cells, CX50P88S does not form gap junctions, but localizes in cytoplasmic multilamellar structures. We studied the dynamics of formation and the stability of these structures in HeLa cells stably transfected with CX50P88S containing a tetracysteine motif appended to its C-terminus (HeLa-CX50P88S(Cys)4 cells). The tetracysteine motif binds the membrane-permeable biarsenical compounds, FlAsH and ReAsH, which become fluorescent upon binding allowing detection of CX50P88S(Cys)4 by fluorescence microscopy or by transmission electron microscopy after the ReAsH-driven fluorescent photoconversion of diaminobenzidine. CX50P88S structures were long-lived. Pulse labeling of HeLa-CX50P88S(Cys)4 cells with FlAsH followed by a chase and ReAsH labeling showed a differential distribution of the labels, with older CX50P88S surrounded by newly synthesized protein. Formation of CX50P88S accumulations was not affected by treatments that block ER-to-Golgi transport. Transmission electron microscopy and tomographic reconstruction revealed that CX50P88S accumulations corresponded to closely apposed circular or semicircular membrane stacks that were sometimes continuous with the rough endoplasmic reticulum. These results suggest that CX50P88S accumulations originate from the rough endoplasmic reticulum and that mutant protein is sequentially added resulting in long-lived cytoplasmic particles. The persistence of these particles in the lens may cause light scattering and the pulverulent cataracts observed in affected individuals.