M174R mutation disrupts GGCX structure and folding. (A) GGCX conservation. Multiple-sequence alignment of GGCX near the identified mutations in 7 species. The amino acid sequences were aligned by CLUSTALW. Black arrows, GGCX mutations identified in the patient; blue arrow, the active site glutamate deprotonation residue H160; red with yellow background, completely conserved residues; yellow with red background, identical residues; white with black background, similar residues; black with white background, different residues. (B) Cell-based carboxylation activity of GGCX and GGCX-GFP fusion protein. (C) Fluorescence confocal microscopy image of GGCX-GFP (top) and GGCX_M174R-GFP fusions (bottom). (D) Immunoblotting of GGCX (lane 1) and the GGCX_M174R (lane 2). Full-length GGCX band is indicated by the arrow.