Interactional cloning of the 84-kDa major outer dense fiber protein Odf84. Leucine zippers mediate associations of Odf84 and Odf27.
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.
Shao
X
X
Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Tarnasky
H A
HA
Schalles
U
U
Oko
R
R
van der Hoorn
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins; D016350:Leucine Zippers; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013082:Sperm Tail / Q000737:chemistry* / Q000648:ultrastructure; D013737:Testis / Q000502:physiology
D003598:Cytoskeletal Proteins; D006360:Heat-Shock Proteins; C490108:Odf1 protein, rat; D011506:Proteins