Figure 4.

Thermolysin-mediated proteolysis of purified or chromatophore membranes embedded R. capsulatus cyt bc₁. (A) Purified cyt bc₁ (0.6 nmol) from the wild type (lanes 1–3) or +2Ala mutant (lanes 4–6) was digested for 1 h with 0.2 nmol of thermolysin in the presence or absence of 20 μM stigmatellin. Aliquots were analyzed by immunoblotting with polyclonal antibodies against the Fe-S subunit of R. capsulatus. Lane 1, wild type (WT), nondigested; lane 2, wild type + thermolysin; lane 3, wild type + thermolysin + stigmatellin; lane 4, +2Ala mutant nondigested; lane 5, +2Ala mutant + thermolysin; lane 6, +2Ala mutant + thermolysin + stigmatellin; lane 7, 18-kDa fragment of the Fe-S subunit as a control (19). (B) Chromatophore membranes (650 μg) from the wild type, +1Ala mutant, or +2Ala mutant were digested for 1 h with 2 nmol thermolysin in the presence or absence of 20 μM stigmatellin. Aliquots were analyzed as described for A; the immunoblot was scanned, and the percentage of the 18-kDa fragment of the Fe-S subunit was calculated and plotted as a bar graph from left to right as wild type + thermolysin, wild type + thermolysin + stigmatellin, +1Ala mutant + thermolysin, +1Ala mutant + thermolysin + stigmatellin, +2Ala mutant + thermolysin, and +2Ala mutant + thermolysin + stigmatellin.