IDENTIFICATION OF A NEW SPORE COAT PROTEIN GENE IN THE CELLULAR SLIME-MOLD DICTYOSTELIUM-DISCOIDEUM

Genomic DNA encoding the prespore cell-specific PL3 cDNA was cloned an d sequenced, revealing that the gene consists of three exons separated by short 100-bp introns. The single long open reading frame predicts a primary translation product of 70 kDa after removal of a cleavable s ignal peptide, two-thirds of which consists of four kinds of amino aci d repeat elements, including two found in other spore coat proteins. T he 85-kDa PL3 protein synthesized in vivo accumulates specifically in regulated secretory vesicles of prespore cells (prespore vesicles), as determined microscopically using antibody against a PL3 gene fusion p roduct expressed in Escherichia coil. The protein later accumulates ex tracellularly in the spore coat, which is formed during sporulation, a s determined ultrastructurally and by Western blot analysis of SDS-PAG E gels. In addition to its high proportion of repeat elements, the PL3 protein has the following properties which distinguish it from other spore coat proteins: (1) it is located at the outer extent of the midd le layer, beneath the outer layer, (2) its dissociation from the coat requires the presence of protein denaturants and reducing agents at el evated temperature, and (3) a large proportion of the protein is not d issociated from the coat even under these conditions, as determined by ultrastructural analysis of the extracted coat. The PL3 protein may c ontribute to the structure of the coat at the interface between the mi ddle, cellulosic layer and the outer, electron-dense, proteinaceous la yer. (C) 1994 Academic Press, Inc.