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 clone TBP-1 (for tat-binding protein-1; designated rat testis TBP-1 [rtTBP-1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP-1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP-1 protein. rtTBP-1 protein contains a heptad repeat of six leucine-type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP-1, consists of a protein triplet with a molecular mass range of 52-48 kDa and acidic pI (5.0-5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin-labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP-1 immunoreactive sites colocalize with alpha-tubulin-decorated manchettes of elongating spermatids. In addition, rtTBP-1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association 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-binding sites-with several subunits of the 26S proteasome, known to be involved in the ATP-dependent degradation of ubiquitin-conjugated proteins.
Rivkin
E
E
Department of Cell Biology and Anatomical Sciences, The City University of New York Medical School, New York 10031, USA.
Cullinan
E B
EB
Tres
L L
LL
Kierszenbaum
A L
AL
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger; D051381:Rats; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013087:Spermatids / Q000378:metabolism; D013091:Spermatogenesis; D013094:Spermatozoa / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C064757:PSMC3 protein, human