TARGETED GENE DISRUPTION REVEALS A ROLE FOR VACUOLIN-B IN THE LATE ENDOCYTIC PATHWAY AND EXOCYTOSIS

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. T he contents of macropinosomes are acidified and digested by lysosomal enzymes, Thereafter, an endocytic marker progresses in an F-actin depe ndent mechanism from the acidic lysosomal phase to a neutral phase, Fr om the post-lysosomal indigestible remnants are released by exocytosis . This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes, Fusions of the vacuolin isof orms to the green fluorescent protein associate with the cytoplasmic s ide of post-lysosomal vacuoles in vivo, Vacuolin isoforms also localis e to patches at the plasma membrane, Since vacuolins have no homologie s to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately, Althoug h the sequences of vacuolins A and B are about 80% identical, only del etion of the vacuolin B gene results in a defect in the endocytic path way; the vacuolin A knock-out appeared to be phenotypically normal, In vacuolin B- mutants endocytosis is normal, but the progression of flu id-phase marker from acidic to neutral pH is impaired, Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in siz e and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.