The role of ubiquitination during the removal of paternal organelles in Caenorhabditis elegans

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Molin, Paola Alexandra
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Middle Tennessee State University
Ubiquitination regulates distinct pathways such as the cell cycle, endocytosis, transcription and DNA repair. We are interested in understanding whether ubiquitination is required for the strict maternal mitochondrial inheritance observed in most metazoans. There are three classes of enzymes responsible for ubiquitination. The ubiquitin activating enzyme is responsible for an ATP-dependent reaction that initially conjugates ubiquitin onto the enzyme. Ubiquitin is then transferred to the ubiquitin conjugating enzyme (UBC), which has a more specific role in determining the type of ubiquitin lysine chain that forms on the substrate. Ubiquitin ligases bind to substrates targeted for ubiquitination. To determine ubiquitin's role in eliminating paternal organelles, we studied the intermediate step in the pathway to determine the UBC responsible for tagging paternal organelles. In nematodes, paternal mitochondria and ubiquitinated sperm derived membranous organelles (MO) are eliminated through autophagy. Using a transgenic C. elegans strain with a GFP::Ubiquitin fusion expressed in the gonad we screened 26 E2s and observed changes in MO ubiquitination. We discovered that ubiquitination on MOs was reduced in simultaneous E2 knockdown of ubc-18 and ubc-16. We used to this system to test the effect reduced ubiquitination had on paternal organelle persistence. Using live cell imaging we discovered that paternal mitochondria persisted during the 2-cell stage in ubc-18 knockdowns. On the other hand, sperm derived MOs also persisted past the 8-cell stage in double E2 knockdowns. ubc-18 drives the recruitment of the proteasome during meiosis I and this localization is important for early removal of paternal mitochondria but not MOs. Ubiquitin is not required for the total elimination of paternal mitochondria. This study provides evidence that a ubiquitin independent pathway is working in conjunction to the ubiquitin dependent pathway. We demonstrate that more than one proteasomal event regulated by ubiquitin receptors RPN-10 and RAD-23 are required for elimination of MOs and paternal mitochondria without disrupting formation of the autophagosomes. Sperm ubiquitination was determined to not play role in tagging paternal organelles, but a possible role of ubiquitination during spermatogenesis and prevention of polyspermy is proposed.