2 PROTEINS OF THE DICTYOSTELIUM SPORE COAT BIND TO CELLULOSE IN-VITRO

The spore coat of Dictyostelium contains nine different proteins and c ellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with u rea and 2-mercaptoethanol could, after removal of urea by gel filtrati on, efficiently bind to particles of cellulose (Avice1), but not Sepha dex or Sepharose. Two proteins, SP85 and SP35, were enriched in the re constitution, and they retained their cellulose binding activities aft er purification by ion exchange chromatography under denaturing condit ions to suppress protein-protein interactions. Neither protein exhibit ed cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino aci d sequencing indicated that SP85 and SP35 are encoded by the previousl y described pspB and psvA genes. This was confirmed for SP85 by showin g that natural M-r polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to recon struct missing elements from the recombinogenic middle region of pspB, SP85 was shown to consist of three sequence domains separated by two groups of the tetrapeptide repeats. Expression of partial pspB cDNAs i n Escherichia coli showed that cellulose-binding activity resided in t he Cys-rich COOH-terminal domain of SP85. This cellulose-binding activ ity can explain SP85's ultrastructural colocalization with cellulose i n vivo. Amino acid composition and antibody binding data showed that S P35 is derived from the Cys-rich N-terminal region of the previously d escribed psvA protein. SP85 and SP35 may link other proteins to cellul ose during coat assembly and germination.