STRUCTURE FUNCTION STUDIES ON THE PH-DEPENDENT ACTIN-BINDING PROTEIN HISACTOPHILIN IN DICTYOSTELIUM MUTANTS/

Our previous studies have shown that the actin-binding protein hisacto philin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner, T o further characterize this interaction we isolated hisactophilin over expression (hisII(+)) and hisactophilin minus (his(-)) mutants, D. dis coideum contains two hisactophilin isoforms; both genes are independen tly transcribed and carry a short intron at the same position of the c oding region, The deduced amino acid sequence of hisactophilin II show ed a characteristic high content of 35 histidine residues out of a tot al 118 amino acids, After transformation of Dictyostelium AX2 wild-typ e cells with a genomic fragment designed to inactivate the hisactophil in I gene we obtained hisactophilin II overexpressing mutants (hisII()). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical pr operties as hisactophilin I during purification and in its pH-dependen t binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high o verexpression, The inactivation of both hisactophilin genes was achiev ed by gene replacement with a vector construct encompassing parts of g ene I and gene II connected by a geneticin cassette, The properties of the hisII(+) and his(-) cells with regard to growth in shaking cultur e and on Klebsiella plates, development, chemotaxis and morphology wer e not affected under normal conditions, However, the hisII(+) transfor mants revealed a significant difference to wild-type cells and his(-) cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor, HisII(+) cells were more resistant to the ac idification; in contrast to AX2 wild-type cells and his(-) cells they did not form plasma membrane protrusions, showed an increase in F-acti n content, and contained large clusters of F-actin, Lowering the inter nal pH caused an accumulation of hisactophilin below the plasma membra ne, The fact that cells deficient in hisactophilin again lose resistan ce to acidification is in good agreement with the hypothesis that hisa ctophilin functions as a pH sensor at the plasma membrane by reversibl y connecting the membrane with the actin cortical network upon local c hanges of the proton concentration.