Figure 1. Matriptase-2 variants processing and plasma membrane localization.

(A) Schematic representation of matriptase-2 (MT2) functional domains and localization of the studied mutations [modified from (Ramsay et al., 2008)]. TM: transmembrane domain. SEA: sea urchin sperm protein, enteropeptidase agrin. CUB: complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 domain. L: low density lipoprotein receptor class A domain. S/P: serine protease domain. The arrow indicates the cleavage activation site. Asterisks indicate the predicted consensus N-glycosylation sites. (B) Quantification of membrane bound matriptase-2 (m-MT2) by binding assay. Hela cells were transiently transfected with the wild type and mutant expressing vectors, or the empty vector, and analyzed for the amount of matriptase-2 on the cell surface. The amount was calculated as the ratio between the absorbance of unpermeabilized and permeabilized cells. Error bars indicate standard deviation. Statistical significance was calculated on a total of three experiments, made in triplicate. Exact P-values are shown above bars. (C) Electron microscopy and morphometric analysis of MT2^wt, MT2^R774C and MT2^MASK. (D) Characterization of MT2. Whole cell extracts and concentrated media of transiently transfected HeLa cells were analyzed by 10% SDS-PAGE. Western blot was performed following standard procedures; MT2 was revealed by the anti-FLAG antibody. s-MT2: soluble MT2. S/P: serine protease domain. CL: cellular lysates; CM: conditioned medium. The equal loading was verified by α-tubulin.