KINETICS OF ENDOSOMAL PH EVOLUTION IN DICTYOSTELIUM-DISCOIDEUM AMEBAS- STUDY BY FLUORESCENCE SPECTROSCOPY

The evolution of endo-lysosomal pH in Dictyostelium discoideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fl uorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an e xtended lag phase lasting for 45-60 min and corresponding to intracell ular transit of FITC-dextran cohort. During the chase period, endosoma l pH decreased during approximately 20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20-40 min to reach p H 6.0-6.2. It was only at this stage that FITC-dextran was released ba ck into the medium with pseudo first-order kinetics. A vacuolar H+-ATP ase is involved in endosomal acidification as the acidification proces s was markedly reduced in mutant strain HGR8, partially defective in v acuolar H+-ATPase and in parent type strain AX2 by bafilomycin A1, a s elective inhibitor of this enzyme. Our data suggest that endocytic car go is channeled from endosomes to secondary lysosomes that are activel y linked to the plasma membrane via recycling vesicles.