ASSEMBLY OF AKAP82, A PROTEIN-KINASE A ANCHOR PROTEIN, INTO THE FIBROUS SHEATH OF MOUSE SPERM

The assembly of the mammalian sperm flagellum is a complex development al event requiring the sequential activation of genes encoding the com ponent parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanis m underlying the assembly of the fibrous sheath surrounding the axonem e was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy d emonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an anti serum was raised against a peptide from the processed region of pro-AK AP82 (M-r 97,000). In immunoblotting experiments, the antibody detecte d pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M-r 109,000 (p109) and M-r 26,000 (p26, representing the ''pro'' domain of the precursor) wer e present in epididymal sperm. Alkaline phosphatase treatment of epidi dymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP8 2 was localized to the entire length of the principal piece in testicu lar sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubiliz ed when germ cells were extracted with Triton X-100. However, in sperm , both AKAP82 and p109 were almost totally resistant to these extracti on conditions and remained in the particulate fraction even after extr action with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of de veloping germ cells. In sperm, much of the RII also became particulate , consistent with the hypothesis that AKAP82 anchors RII in the flagel lum. These data indicate that pro-AKAP82 is synthesized in the cell bo dy, transported down the axoneme to its site of assembly in the fibrou s sheath, and then proteolytically clipped to form mature AKAP82. (C) 1997 Academic Press.