Abstract
Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis (Arabidopsis thaliana). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation.
Highlights
Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes
We focused on the requirement of chloroplast-associated E3s for the induction of chloroplast-targeting autophagy in Arabidopsis and showed that PLANT U-BOX4 (PUB4) and SUPPRESSOR OF PPI1 LOCUS1 (SP1) are dispensable for the induction of both chlorophagy and Rubiscocontaining bodies (RCBs)-mediated autophagy
We began our assessment of the requirement for PUB4 in the induction of chlorophagy by using a mutant allele of Arabidopsis PUB4, pub4-6 (Supplemental Fig. S1A), in which an amino acid substitution in PUB4 compromises the ubiquitination of reactive oxygen species (ROS)-overaccumulating chloroplasts (Woodson et al, 2015)
Summary
Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. By contrast, during the major type of autophagy, known as macroautophagy, nascent, double membranebound vesicles (termed autophagosomes) engulf a portion of the cytoplasm including proteins and organelles, which allows the bulk digestion of a subset of intracellular components (Mizushima and Komatsu, 2011) This degradation system is vital in enabling cells to adapt to starvation through the efficient recycling of cytoplasmic components. One well-characterized microautophagy process is the degradation of peroxisomes in the methylotrophic yeast Komagataella phaffii (previously known as Pichia pastoris) In this micropexophagy process, core ATG proteins are required for the formation of a specific membrane structure to complete the sequestration of targeted peroxisomes (Mukaiyama et al, 2004)
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