Structural features of the 26S proteasome complex isolated from rat testisand sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the r at spermatid manchette and outer dense fibers of the developing sperm. TBP- 1 contains a heptad repeat of six-leucine zipper fingers at the amino termi nus and highly conserved ATPase and DNA/RNA helicase motifs toward the carb oxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory c omplex 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 electr on microscopy using negative staining. The 26S proteasome from rat testis w as fractionated by Sephactyl 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 t he 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 abou t 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alpha beta beta alpha), capp ed by two polar 19S regulatory complexes, each identified by an element kno wn as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subuni t of the 19S regulatory cap. Rat sperm preparations displayed both dissocia ted 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasome s in the perinuclear-arranged manchette are in a suitable location for reco gnition, sequestration, and degradation of accumulating ubiquitin-conjugate d 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. Mo l. Reprod. Dev. 57:176-184 2000. (C) 2000 Wiley-Liss, Inc.