SELF-INTERACTION OF THE MAJOR 27-KILODALTON OUTER DENSE FIBER PROTEINIS 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 amin o acid structure had indicated the presence of a putative leucine zipp er dimerization motif in the N-terminus and the presence of PCX repeat s in the C-terminus. We had previously shown that the ODF27 N-terminal fragment can interact with full-length ODF27. We have used two differ ent methods to analyze this interaction further. First we used fusion proteins between glutathione S-transferase (GST) and ODF27-derived fra gments 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 o f GST and the ODF27 leucine zipper did not interact with ODF27. We fou nd that the ODF27 C-terminal half can also interact with ODF27. The ye ast two-hybrid method nas 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 abolishes this interaction, and 3) the PCX repeats are involved in the self-interaction of the ODF27 C-t erminus. The detected self-associations are weak. To analyze the molec ular weight of in vitro-translated ODF27, we carried out gel filtratio n experiments. They show that at low concentrations, a fraction of ODF 27 proteins exists as multimers while the rest are monomers whose shap e deviates considerably from that of globular proteins. Our results id entify regions in the N- and C-termini of ODF27 involved in self-inter actions and suggest that in ODF, where high protein concentrations pre vail, ODF27 can self-interact.