FIGURE 5.

Pulse-chase analysis of MT1-MMP. COS cells were transfected with HA-tagged MT1-MMP constructs, metabolically labeled with l-[S³⁵]methionine for 25 min, and chased for the indicated intervals. a, comparable expression levels of MT1-MMP constructs were confirmed by immunoblotting with monoclonal anti-HA Ab. b, immunoprecipitates of MT1-MMP with monoclonal anti-HA Ab were analyzed by SDS-PAGE and autoradiography. The combination of the results from the metabolic pulse-chase with the glycosylation analysis of MT1-MMP by site-directed mutagenesis and enzymatic desialylation (compare Fig. 2, b and c) allow the assignment of the protein bands to distinct processing steps. In brief, these steps include: 1, synthesis of the protein; 2, glycosylation (absent in CHO); 3 and 4, activation with loss of the pro-domain; and 5 and 6, generation of a membrane-tethered, autocatalytic cleavage product (see “Results” and “Discussion” for further details) (control = untransfected COS; * = transfected controls without precipitating Ab).