No abstract

Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants.

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (–Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of –Pi or salt stressed plants, or senescing leaves. Transiently expressed AtPAP17-GFP localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss-of-function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked upregulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH-activity profile, and rapid repression and turnover following Pi-resupply to –Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt stressed or –Pi atpap17 mutant, do not support proposals that AtPAP17’s peroxidase activity contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 upregulation.

No abstract

Autophagy is a highly conserved pathway from yeast to human, and a basic catabolic mechanism that degrades unnecessary or dysfunctional cellular components. Recently studies have shown that autophagy is involved in stem cell homeostatic control during aging, regeneration and tissue reprogramming. It has been shown that autophagy is required for self‐renewal and differentiation of adult human stem cells. While the function of autophagy in planarian stem cells and regeneration has not been clearly demonstrated yet. To decipher if autophagy is required for planarian regeneration, we identified the homologs of several yeast Atg proteins from our transcriptome database of Dugesia japonica. We found three planarian homologs of yeast Atg8, and named them DjAtg8A, DjAtg8B, and DjAtg8C. In situ hybridization of DjAtg8 paralogs showed that all three genes are expressed in intestine, suggesting their conserved function in endocytosis and lysosomes. In addition, γ‐irradiation induced the up‐regulation of DjAtg8s expression and the lipidation of DjAtg8A, indicates the autophagy pathway is conserved and activated in planarian under stress. Our preliminary data showed different phenotypes in planarians with depletion of various DjAtg8 genes, and provided evidence for the putative function of Atg8 proteins in planarian digestion system and response to stress, instead of regeneration.

Under oxidative stresses, accumulation of misfolded protein in the endoplasmic reticulum (ER) activates downstream pathway ‐ the unfolded protein response (UPR) to protect cells. Autopahgy, on the other hand, is another protective response to cope with cellular stresses in eukaryotes, which involves in the degradation of cytosolic proteins and defective organelles. It is known that the ER‐selective autophagy, or ERphagy, will be induced during ER stresses and serve a protective role to maintain cell survival. However, the correlation of ERphagy with other type of autophagy is not clear. Here, we show that ERphagy is a selective process distinct from the cytoplasm‐to‐vacuole targeting (Cvt) pathway and the non‐selective macroautophagy. We found that under the dithiothreitol (DTT)‐induced ER stress, the ER‐containing autophagosomes (ERAs) would tend to accumulate in cytoplasm rather than fuse with vacuoles rapidly. However, soon after DTT is removed, ERAs will start to fuse with vacuoles and be degraded. We also discovered that the Cvt pathway would be partially inhibited at the vesicle forming stage during ERphagy, causing the delayed maturation phenotype of prApe1. Nevertheless, ERphagy seems to have limited effect on the process of the starvation–induced macroautophagy, and starvation treatment would not promote the clearance of accumulated ERAs in cytoplasm either. Taken these together, ERphagy as a selective process may compete with the Cvt pathway for utilizing the share of common regulatory machinery, but not preferable to the starvation‐induced macroautophagy.NSC96‐2311‐B‐002‐012‐MY3

Autophagy, as a conserved pathway in eukaryotic cells, plays a critical role in maintaining cellular homeostasis by delivering cytoplasmic materials to lysosomes/vacuoles for degradation. Abnormal autophagy activities are associated with several human diseases, such as diabetes, neurodegenerative diseases, and cancers. These observation leads to the possibilities of manipulating autophagy for therapeutic applications. To achieve this purpose, detailed information on the molecular mechanisms of autophagy regulation is essential. In this study, we have developed a protocol to screen for more selective autophagy regulatory genes based on cell image analysis, which is compliable to cell sorter application. We constructed a plasmid for expressing peroxisome‐targeted pH‐sensitive green fluorescent proteins in Saccharomyces cerevisiae. The plasmid was introduced into yeast deletion mutant pools and pexophagy‐deficient cells with high fluorescent signals after induction of pexophagy was sorted out. The identities of the mutated genes of those selected cells were determined by next generation sequencing of the gene barcodes. Our results proved it is able to distinguish atg mutants from wild types cells. We are currently characterizing the isolated autophagy mutants from the nonessential yeast gene deletion mutant pool. Grant support: NSC 101–2311‐B‐002–005