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Structural features of the 26S proteasome complex isolated from rat testis and sperm tail
Author(s) -
Mochida Kazuhiko,
Tres Laura L.,
Kierszenbaum Abraham L.
Publication year - 2000
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/1098-2795(200010)57:2<176::aid-mrd9>3.0.co;2-o
Subject(s) - biology , proteasome , spermatid , sperm , protein subunit , microbiology and biotechnology , negative stain , leucine zipper , aaa proteins , atpase , biochemistry , peptide sequence , electron microscope , enzyme , genetics , physics , optics , gene
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 (αββα), 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. Mol. Reprod. Dev. 57:176–184, 2000. © 2000 Wiley‐Liss, Inc.