IDENTIFICATION AND CHARACTERIZATION OF ESPIN, AN ACTIN-BINDING PROTEIN LOCALIZED TO THE F-ACTIN-RICH JUNCTIONAL PLAQUES OF SERTOLI-CELL ECTOPLASMIC SPECIALIZATIONS

Ectoplasmic specializations are membrane-cytoskeletal assemblages foun d in Sertoli cells at sites of attachment to elongate spermatids or ne ighboring Sertoli cells, They are characterized in part by the presenc e of a unique junctional plaque which contains a narrow layer of paral lel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum, Using a monoclonal antibody, we have identified 'espin,' a novel actin-binding protein l ocalized to ectoplasmic specializations. By immunogold electron micros copy, espin was localized to the parallel actin bundles of ectoplasmic specializations at sites where Sertoli cells contacted the heads of e longate spermatids. The protein was also detected at the sites of ecto plasmic specializations between neighboring Sertoli cells, Espin exhib its an apparent molecular mass of similar to 110 kDa in SDS gels, It i s encoded by an similar to 2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined, On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein w hich contains 8 ankyrin-like repeats in its N-terminal third, a potent ial P-loop, two proline-rich peptides and two peptides which contain c lusters of multiple glutamates bracketed by arginines, lysines and glu tamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin, The ankyrin-like repeats and a 66 amino acid pep tide in the C terminus show significant sequence similarity to protein s encoded by the forked gene of Drosophila. A fusion protein containin g the C-terminal 378 amino acids of espin was found to bind with high affinity (K-d=similar to 10 nM) to F-actin in vitro with a stoichiomet ry of similar to 1 espin per 6 actin monomers, When expressed by trans fected NRK fibroblasts, the same C-terminal fragment of espin was obse rved to decorate actin fibers or cables, On the basis of its structure , localization and properties, we hypothesize that espin is involved i n linking actin filaments to each other or to membranes, thereby poten tially playing a key role in the organization and function of the ecto plasmic specialization.