A linking function for the cellulose-binding protein SP85 in the spore coat of Dictyostelium discoideum.
SP85 is a multidomain protein of the Dictyostelium spore coat whose C-terminal region binds cellulose in vitro. To map domains critical for localizing SP85 and for binding to other proteins in vivo, its N- and C-terminal regions, and a hybrid fusion of the N- and C-regions, were expressed in prespore cells. Immunofluorescence showed that only the N-terminal region and the N/C-hybrid accumulated in prespore vesicles, where coat proteins are normally stored prior to secretion. In contrast, only the C-terminal region and N/C-hybrid were incorporated into the coat after secretion. To determine if SP85 is important for the incorporation of other coat proteins, an SP85-null strain was created and found to mislocalize the coat protein SP65 to the interspore matrix. In vitro binding studies demonstrated that the SP85 C-terminal region bound SP65 and cellulose simultaneously, and SP65 incorporation was rescued in vivo by the C-terminal region. SP85-null spores showed increased latent permeability to a fluorescent lectin probe and accelerated germination times, and decreased buoyant density of their coats, suggesting that coat barrier functions were compromised. Dominant negative reductions in barrier functions also resulted from expression of the SP85 terminal regions, suggesting that a linking activity was important for SP85's function. Thus, separate domains of SP85 specify prespore vesicle compartmentalization and coat incorporation, and additional domains link SP65 to the coat and simultaneously interact with other binding partners which contribute to coat barrier functions.
Zhang
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Department of Anatomy, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA.
Zhang
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West
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CM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology*; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013170:Spores
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D015800:Protozoan Proteins; C113756:PspB protein, Dictyostelium discoideum; D002482:Cellulose