Extracellular ubiquitin system implicated in fertilization of the ascidian, XXXX : isolation and characterization.

@entity3282 @entity855 @entity653 @entity25598
The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, @entity25598 . Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by @entity653 density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires @entity3282 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, @entity855 and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with @entity855 and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.
