Mechanism of extracellular ubiquitination in the mammalian epididymis

Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E1 and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.