Ubiquitination is required for the initial removal of paternal organelles in C. elegans

Abstract

Elimination of paternal mitochondria after fertilization occurs in many species using the process of selective autophagy. The mechanism for targeting paternal mitochondria, but not maternal mitochondria, for elimination in the early embryo is not well understood. The results in this paper suggest that there are at least two different mechanisms for targeting paternal mitochondria for elimination: the first involving ubiquitination and a second involving a mitochondrial associated autophagy receptor, fndc-1. Elimination of paternal mitochondria can be visualized in embryos of the nematode, C. elegans. Paternal mitochondria enter the zygote at fertilization. Initially, they are closely associated with another sperm organelle, the membraneous organelle (MO). The MOs become ubiquitinated within minutes after fertilization. Simultaneous RNAi knockdown of two ubiquitin conjugating enzymes, ubc-18 and ubc-16, reduces MO ubiquitination. Loss of function of ubc-18 alone leads to loss of K48-linked polyubiquitin chains and halts the recruitment of proteasome to MOs. Interestingly, knockdown of ubc-18 or ubc-16 or the combination does not reduce the localization of K63-linked ubiquitin chains to MOs suggesting that some ubiquitin structure other than K63 chains is responsible for recruiting the autophagy machinery to MOs. Double knockdown (ubc-18/ubc-16) inhibits the recruitment of the autophagy protein, LGG-1 (homolog of LC3/GABARAP), to paternal organelles and causes the persistence of paternal mitochondria into the two cell stage. If paternal mitochondria are not eliminated via this early process, they are eventually removed from the embryo in a process that depends on the mitophagy adaptor protein, fndc-1. Thus, there are two redundant, but temporally distinct mechanisms that target paternal mitochondria for elimination in C. elegans. In addition to the involvement of ubiquitination in the elimination of paternal mitochondria, two subunits of the proteasome, rpn-10 and rad-23, are required for elimination of paternal mitochondria. These subunits are known to function as ubiquitin receptors and knockdown of either inhibits the recruitment of proteasome to ubiquitinated MOs. Their knockdown does not affect the localization of LGG-1 to paternal structures indicating that the proteasome is not required for autophagy membrane recruitment but might be involved in autophagosome maturation or its fusion with the lysosome.