Two proteins of the Dictyostelium spore coat bind to cellulose in vitro.
The spore coat of Dictyostelium contains nine different proteins and cellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with urea and 2-mercaptoethanol could, after removal of urea by gel filtration, efficiently bind to particles of cellulose (Avicel), but not Sephadex or Sepharose. Two proteins, SP85 and SP35, were enriched in the reconstitution, and they retained their cellulose binding activities after purification by ion exchange chromatography under denaturing conditions to suppress protein--protein interactions. Neither protein exhibited cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino acid sequencing indicated that SP85 and SP35 are encoded by the previously described pspB and psvA genes. This was confirmed for SP85 by showing that natural M(r) polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to reconstruct 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 in Escherichia coli showed that cellulose-binding activity resided in the Cys-rich COOH-terminal domain of SP85. This cellulose-binding activity can explain SP85's ultrastructural colocalization with cellulose in vivo. Amino acid composition and antibody binding data showed that SP35 is derived from the Cys-rich N-terminal region of the previously described psvA protein. SP85 and SP35 may link other proteins to cellulose during coat assembly and germination.
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, USA.
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Y
Zhang
R D
RD
Brown
C M
CM
West
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D002480:Cellulase / Q000302:isolation & purification; D002482:Cellulose / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013172:Spores, Fungal / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D018076:DNA, Complementary; D005656:Fungal Proteins; D015800:Protozoan Proteins; C113756:PspB protein, Dictyostelium discoideum; D011994:Recombinant Proteins; C067492:prespore protein EB4-PSV, Dictyostelium discoideum; C061925:spore coat proteins, Dictyostelium; D002482:Cellulose; D002480:Cellulase