TLRR (Irrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis

Background information. Spermatozoa are formed via a complex series of cellular transformations, including acrosome and flagellum formation, nuclear condensation and elongation and removal of residual cytoplasm. Nuclear elongation is accompanied by the formation of a unique cytoskeletal structure, the manchette. We have previously identified a leucine-rich repeat protein that we have named TLRR (testis leucine-rich repeat), associated with the manchette that contains a PP1 (protein phosphatase-l)-binding site. Leucine-rich repeat proteins often mediate protein-protein interactions; therefore, we hypothesize that TLRR acts as a scaffold to link signalling molecules, including PP1, to the manchette near potential substrate proteins important for spermatogenesis. Results. TLRR and PP1 interact with one another as demonstrated by co-immunoprecipitation and the yeast two-hybrid assay. TLRR binds more strongly to PP1 gamma 2 than it does to PP1 alpha. Anti-phosphoserine antibodies immunoprecipitate TLRR from testis lysate, indicating that TLRR is a phosphoprotein. TLRR is part of a complex in testis that includes cytoskeletal proteins and constituents of the ubiquitin-proteasome pathway. The TLRR complex purified from 3T3 cells contains similar proteins, co-localizes with microtubules and is enriched at the microtubule-organizing centre. TLRR is also detected near the centrosome of elongated, but not mid-stage, spermatids. Conclusion. We demonstrate here that TLRR interacts with PP1, particularly the testis-specific isoform, PP1 gamma 2. Immunoaffinity purification confirms that TLRR is associated with the spermatid cytoskeleton. In addition, proteins involved in protein stability are part of the TLRR complex. These results support our hypothesis that TLRR links signalling molecules to the spermatid cytoskeleton in order to regulate important substrates involved in spermatid transformation. The translocation of TLRR from the manchette to the centrosome region suggests a possible role for this protein in tail formation. Our finding that TLRR is associated with microtubules in cultured cells suggests that TLRR may play a common role in modulating the cytoskeleton in other cell types besides male germ cells.