Structural features of the 26S proteasome complex isolated from rat testis and sperm tail.
We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.
Mochida
K
K
Department of Cell Biology and Anatomical Sciences, The City University of New York Medical School, New York, NY 10031, USA.
Tres
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Kierszenbaum
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D000818:Animals; D046911:Macromolecular Substances; D008297:Male; D008854:Microscopy, Electron; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D013082:Sperm Tail / Q000201:enzymology*; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000201:enzymology*
D046911:Macromolecular Substances; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease