Interactional Cloning of the 84-kDa Major Outer Dense Fiber Protein Odf84

Abstract

The study of mammalian sperm tail outer dense fibers (ODF), a structure of unknown function, is hampered by the insoluble nature of ODF proteins and the availability of only one cloned component, Odf27. We report here the first use of the Odf27 leucine zipper as bait in a yeast two-hybrid screen to isolate a novel testis-specific protein whose interaction with Odf27 depends critically on the Odf27 leucine zipper. We find that the novel gene, 111-450, encodes a product that localizes to ODF as determined by fluorescence microscopy and immunoelectron microscopy and that the gene 111-450 product is identical to the major ODF protein, Odf84. Interestingly, Odf84 contains two C-terminal leucine zippers, and we demonstrate that all leucine residues in the upstream leucine zipper are required for interaction with Odf27, demonstrating the strategic validity of our approach. The use of the yeast screening approach to isolate leucine zipper containing proteins should be useful in other systems, and our findings have implications for ODF structural models.

A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality

“…These findings are in contrast with earlier studies of Odf2 expression, which identified it as being testis specific (Brohmann et al, 1997;Hoyer-Fender et al, 1998;Schalles et al, 1998;Shao et al, 1997;Turner et al, 1997). Odf2 was reportedly expressed specifically in male germ cells from the secondary spermatocyte stage onwards, with expression reaching its maximal level in the round spermatid (Horowitz et al, 2005;Hoyer-Fender et al, 1998;Turner et al, 1997).…”

“…As a result, details of the in vivo role of ODF2 in formation of the outer dense fibers and in its recently identified role at the centrosome have been lacking. Such a model would be invaluable in determining whether Odf2 is required specifically during spermatogenesis, as was originally suggested (Brohmann et al, 1997;Hoyer-Fender et al, 1998;Schalles et al, 1998;Shao et al, 1997;Turner et al, 1997), or whether Odf2 also plays key roles at the centrosome in vivo.…”

“…Early reports consistently reported murine Odf2 expression as testis specific, as determined by Northern blotting (Brohmann et al, 1997;Hoyer-Fender et al, 1998;Schalles et al, 1998;Shao et al, 1997;Turner et al, 1997). However, in a recent publication, monoclonal antibodies directed against purified chicken centrosomes have identified ODF2 protein as a widespread centrosomal component (Nakagawa et al, 2001).…”

“…Outer dense fiber 2 (ODF2) protein was identified as a major component of the outer dense fibers of the sperm flagellum (Brohmann et al, 1997;Schalles et al, 1998;Shao et al, 1997;Turner et al, 1997). Nine outer dense fibers surround the microtubules of the axoneme and although they may not contribute directly to motility, they are believed to maintain the elastic properties of the sperm tail and protect it from shear stress during epididymal transport and ejaculation (Baltz et al, 1990).…”

“…ODF2 is a putative coiled coil protein containing two leucine zippers that mediate interaction with ODF1, another major component of the outer dense fibers (Donkor et al, 2004;Shao et al, 1997). A critical function of Odf2 is implied by strong sequence conservation (Petersen et al, 1999): for example, the amino acid sequences of human and mouse ODF2 share greater than 97% homology.…”