FIG 3.

Tat-dependent membrane association of NiFe-cofactor-free Hyd-2. (A) Western blot analysis using anti-Hyd-2 antiserum of crude extracts (30 μg protein) from CP695 (ΔhybC) or strain DADE (ΔtatA-E [deletion of tatA through tatE genes]) transformed either with a plasmid encoding pro-HybC or with a plasmid encoding artificially matured HybC_proc. SF, soluble fraction; MF, membrane fraction. The asterisk denotes an unidentified membrane-associated cross-reacting polypeptide species. The migration positions of pro-HybC and HybC (equivalent to HybC_proc) have been generically labeled HybC because the respective bands were not distinguished in the experiment. The migration position of mature HybO is shown. (B) Western blot analysis of crude extracts (30 μg protein) from anaerobically grown strain CB10 (ΔhypF ΔhybC) transformed with a plasmid encoding either pro-HybC or genetically matured HybC_proc. Detection was performed using anti-Hyd-2 antiserum. The first lane shows a membrane fraction (MF) derived from strain FTD147, which lacks the large subunit of Hyd-1, Hyd-2, and Hyd-3. SF, soluble fraction. The asterisk denotes an unidentified cross-reacting species.