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Fig. 5. The transmembrane domain is required for the trafficking of MT-MMP to the cell surface of CHO cells. (A) Domain structure of mutant MT1-MMP. PRO, prodomain; CAT, catalytic domain; H, hinge region; PEX, hemopexin domain; TM, transmembrane domain; CT, cytoplasmic tail. (B) Immunoblotting and gelatin zymography of CHO cells expressing mutant MT1-MMP. Cells were lysed in a RIPA buffer containing a proteinase inhibitor cocktail-Set III and phenylmethylsulfonyl fluoride (Calbiochem, San Diego, CA, USA). The cell lysates were analyzed by western blotting with the MT1-MMP antibody Ab815 (upper panel). The ability of cells to activate MMP-2 was analyzed by gelatin zymography (bottom panel). An arrowhead indicates the gelatinolytically active, soluble, short MT-TM/CT construct found in the medium. Self-proteolysis of MT1-MMP leads to this short species (Osenkowski et al., 2004; Rozanov and Strongin, 2003). (C) Confocal microscopy of CHO cells expressing mutant MT1-MMP. Permeabilized and non-permeabilized cells were stained with the Ab815 antibody. The XY and ZX projections of cells are shown at the bottom and on the right of each panel, respectively. Notice the absence of staining of the non-permeabilized TM/CT cells.

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