Morphometric analysis and processing of MT2 variants. (A) Schematic representation of MT2 functional domains and localization of the studied mutations. TM indicates 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. *Predicted consensus N-glycosylation sites. (B) Electron microscopy and (C) morphometric analysis of MT2 variants. HeLa cells were transiently transfected with Lipofectamine 2000 using pcDNA3.1 expressing WT and mutant MT2. After 18 hours, cells were fixed, labeled with a polyclonal rabbit anti-FLAG using the gold-enhance protocol, embedded in Epon-812, and cut. Immunoelectron microscopy (EM) images were acquired from thin sections under a Philips Tecnai-12 electron microscope (Philips, Eindhoven, The Netherlands) using an ULTRA VIEW CCD digital camera (Philips). Images were acquired using AnalySIS software (Soft Imaging System, Lakewood, CO; original magnification ×23 000; B). Thin sections were used to quantify gold particles residing within different compartments of the secretory pathway (C). PM indicates plasma membrane; ER, endoplasmic reticulum; and G, Golgi. (D) Characterization of wild-type and mutant MT2. Whole-cell extracts and concentrated media of transiently transfected HeLa cells were analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blot was performed following standard procedures; MT2 was revealed by the anti-FLAG antibody. CL indicates cellular lysates; and CM, conditioned medium. The equal loading was verified by antitubulin. Scales refer to relative molecular mass (in kilodaltons).