A PROTEIN ASSOCIATED WITH THE MANCHETTE DURING RAT SPERMIOGENESIS IS ENCODED BY A GENE OF THE TBP-1-LIKE SUBFAMILY WITH HIGHLY CONSERVED ATPASE AND PROTEASE DOMAINS

We have used a rat pachytene spermatocyte cDNA expression library to c lone TBP-1 (for tat-binding protein-1; designated rat testis TBP-1 [rt TBP-1]), a new member of the family of putative ATPases associated wit h the 26S proteasome complex. The 1.63 kb rtTBP-1 cDNA encodes a 49 kD a protein with 99% amino acid identity to human TBP-1 protein, rtTBP-1 protein contains a heptad repeat of six leucine-type zipper fingers a t the amino terminal end and highly conserved ATPase and DNA/RNA helic ase motifs towards the carboxyl terminal region. Chromato-focusing fra ctionation of rat testis sucrose extracts demonstrates that the encode d product, recognized by an antiserum raised to the first 196 amino ac ids of human TBP-1, consists of a protein triplet with a molecular mas s range of 52-48 kDa and acidic pl (5.0-5.9). An identical immunoreact ive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In si tu hybridization using digoxigenin-labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithe lial region occupied by elongating spermatids and primary spermatocyte s. Indirect immunofluorescence and immunogold electron microscopy stud ies show that rtTBP-1 immunoreactive sites colocalize with a-tubulin-d ecorated manchettes of elongating spermatids. In addition, rtTBP-1 imm unoreactivity was detected in fibrillar and granular cytoplasmic bodie s typically observed in spermatocytes and spermatids as well as in ass ociation with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP-1 is a member of the highly evolutionary conserved TBP-1-like subfamily of putative ATPases, sharing regions of identity-including ATP-bindin g sites-with several subunits of the 26S proteasome, known to be invol ved in the ATP-dependent degradation of ubiquitin-conjugated proteins. (C) 1997 Wiley-Liss, Inc.