Self-interaction of the major 27-kilodalton outer dense fiber protein is in part mediated by a leucine zipper domain in the rat.
The RT7 gene is exclusively expressed in spermatids and encodes the 27-kDa major outer dense fiber (ODF) protein ODF27. Analysis of its amino acid structure had indicated the presence of a putative leucine zipper dimerization motif in the N-terminus and the presence of PCX repeats in the C-terminus. We had previously shown that the ODF27 N-terminal fragment can interact with full-length ODF27. We have used two different methods to analyze this interaction further. First we used fusion proteins between glutathione S-transferase (GST) and ODF27-derived fragments to show that the N-terminal half of ODF27 as well as the first 100 amino acids can interact with ODF27. A fusion protein consisting of GST and the ODF27 leucine zipper did not interact with ODF27. We found that the ODF27 C-terminal half can also interact with ODF27. The yeast two-hybrid method was next employed to analyze these interactions in vivo. We found that 1) N-terminal fragments containing the leucine zipper interact with the ODF27 N-terminus, but not with its C-terminus, 2) deletion of the leucine zipper abolished this interaction, and 3) the PCX repeats are involved in the self-interaction of the ODF27 C-terminus. The detected self-associations are weak. To analyze the molecular weight of in vitro-translated ODF27, we carried out gel filtration experiments. They show that at low concentrations, a fraction of ODF27 proteins exists as multimers while the rest are monomers whose shape deviates considerably from that of globular proteins. Our results identify regions in the N- and C-termini of ODF27 involved in self-interactions and suggest that in ODF, where high protein concentrations prevail, ODF27 can self-interact.
Shao
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Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
van der Hoorn
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D000818:Animals; D002850:Chromatography, Gel; D019281:Dimerization; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006360:Heat-Shock Proteins; D016350:Leucine Zippers; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics
D006360:Heat-Shock Proteins; C490108:Odf1 protein, rat; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase