PRODIGIOSINS UNCOUPLE LYSOSOMAL VACUOLAR-TYPE ATPASE THROUGH PROMOTION OF H+ CL- SYMPORT/

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takat suki, Kondo, Yamasaki and Nagai (1995) FEES Lett, 359, 53-59] that pro digiosin 25-C (one of the red pigments of the prodigiosin group produc ed by micro-organisms like Streptomyces and Serratia) uncoupled vacuol ar H+-ATPase, inhibited vacuolar acidification and affected glycoprote in processing. In the present study we show that prodigiosin, meta-cyc loprodigiosin and prodigiosin 25-C, all raise intralysosomal pH throug h inhibition of lysosomal acidification driven by vacuolar-type (V-)AT Pase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lyso somal acidification was quick and reversible, showing kinetics of simp le non-competitive (for ATP) inhibition. However, the prodigiosins nei ther raised the internal pH of isolated lysosomes nor showed ionophori c activity against H+ or K+ at concentrations where they strongly inhi bited lysosomal acidification. They required Cl- for their acidificati on inhibitory activity even when driven in the presence of K+ and vali nomycin, suggesting that their target is not anion (chloride) channel( s). In fact, the prodigiosins inhibited acidification of proteoliposom es devoid of anion channels that were reconstituted from lysosomal vac uolar-type (V-)ATPase and Escherichia call phospholipids. However, the y did not inhibit the formation of an inside-positive membrane potenti al driven by lysosomal V-ATPase. Instead, they caused quick reversal o f acidified pH driven by lysosomal V-ATPase and, in acidic buffer, pro duced quick acidification of lysosomal pH, both only in the presence o f Cl-. In addition, they induced swelling of liposomes and erythrocyte s in iso-osmotic ammonium salt of chloride but not of gluconate, sugge sting the promotion of Cl- entry by prodigiosins. These results sugges t that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.