During meiotic cell division, homologous chromosomes must pair and undergo crossover recombination, producing a physical linkage that promotes proper chromosome segregation. Recombination is accompanied by a structural reorganization of chromosomes around the crossover site to facilitate segregation on the Meiosis I spindle. In C. elegans, ZHP-3 is required for both crossover formation and for the proper restructuring of chromosomes in late prophase (Jantsch et al., 2004; Bhalla et al., 2008). The presence of a conserved RING finger motif in ZHP-3 suggested that it might act as a ubiquitin E3 ligase and modify substrates to alter their stability, localization, and/or activity. To characterize the ubiquitin ligase activity of ZHP-3, several experiments have been conducted. First, ZHP-3 purified from bacteria shows auto-ubiquitination activity. In addition, a ZHP-3 transgenic lines was generated by microparticle bombardment to facilitate purification of ZHP-3 from adult worms. The construct was designed with
zhp-3 genomic sequence under control of the
pie-1 promoter, with an N-terminal mCherry-TEV-S (mCherry::
zhp-3) tag. The mCherry::
zhp-3 transgene can fully rescue the phenotype of the
zhp-3(
jf61) null allele, recapitulating the localization pattern of the endogenous protein, localizing along the synaptonemal complex in pachytene and as foci in diplotene. The transgenic line displays wildtype levels of viability at 15deg and 20deg C. At 25deg C, there is a slight decrease in viability and an increase in the frequency of male self-progeny, which may be due to elevated expression of the transgene at higher temperatures. The mCherry::
zhp-3 strain undergoes crossover recombination, as indicated by the presence of 6 DAPI-staining bivalents at diakinesis. Bivalent chromosomes are properly structured, with HTP-1 localization restricted to the long arm of the bivalent. These data indicate that the mCherry::
zhp-3 transgene is competent to carry out both of ZHP-3's roles. In agreement with experiments performed with recombinant protein, mCherry-ZHP-3 immunopurified from adult worm lysate had ubiquitination activity. Mass spectrometry analysis will be performed to identify proteins that co-immunopurify with mCherry-ZHP-3, including targets of its ubiquitination activity. Additionally, candidate substrates will be in vitro transcribed and translated and included in the in vitro ubiquitination reaction. Together, these data help elucidate how ZHP-3's ubiquitin ligase activity facilitates the large-scale changes in chromosome structure in late prophase that are required for proper meiotic chromosome segregation.