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Translation inhibition is a major but poorly understood mode of action of micro(mi)RNAs in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here we show that the translation inhibition but not the mRNA cleavage activity of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild type and the amp1 mutant for membrane-bound but not total polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain-containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2-green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum-derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis.

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H(+)-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process. T he endosomal-lysosomal/vacuolar pathway is the primary catabolic system of eukaryotic cells that degrades extracellular and intracellular materials. Membrane proteins destined for degradation, such as misfolded proteins or endocytosed receptors, become tagged by ubiquitin for further sorting to the endosomal-lysosomal/vacuolar system for degradation (1). During this process, an evolutionarily conserved machinery called endosomal sorting complex required for transport (ESCRT), is responsible for sorting these ubiquitinated cargos into the intraluminal vesicles (ILVs) of prevacuolar compartments/multivesicular bodies (PVCs/MVBs), which subsequently fuse with vacuoles/lysosomes to deliver their contents into the lumen for proteolytic degradation (2, 3). Malfunction of the assembly or dissociation of the ESCRT machinery disrupts MVB formation and thus results in the accumulation of ubiquitinated membrane cargos (4, 5).Macroautophagy (hereafter as autophagy) is another highly conserved catabolic process, which converges on the endosomallysosomal/vacuolar pathway to deliver aberrant organelles, longlived proteins, and protein aggregates to the lysosome/vacuole via a unique structure termed the "autophagosome" (6). Morphologically different from MVBs, autophagosomes are characterized by a double membrane structure, which is initiated from the phagophore assembly site/preautophagosome site (PAS) (7). The proteins or organelles to be degraded are encapsulated by autophagosomes that fuse either directly with the vacuole/lysosome or with endosomes like MVBs for expansion/maturation to form amphisomes, which then fuse with vacuole/lysosome for degradation. A number of conserved autophagy-related gene (ATG) proteins have been identified as participating in the autophagy pathway in eukaryotic cells (8).Even though it is generally accepted that at least one population of...

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphatedependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.O ne long-lasting question regarding autophagosome biogenesis is its membrane origin (1). The initiation site for autophagosomes is termed the preautophagosomal structure or phagophore assembly site (PAS). However, the source of the phagophore membrane remains controversial in different systems, and exactly how the phagophore is initiated from its membrane origin is still unclear. The core autophagy-related (ATG) machinery regulates phagophore assembly in a spatiotemporally coordinated manner whereas some of the ATG components will disassociate from the completed autophagosome and some are turned over together with the autophagosome (1-3).As the sole transmembrane protein, autophagy-related protein 9 (ATG9) has long been suggested to provide a lipid/membrane source for autophagosome formation because ATG9-deficient mutants in yeast or mammal fail to form autophagosomes (4, 5). Although ATG9 is conserved in all eukaryotes (6), it seems that ATG9 might perform its function divergently in different systems. In yeast, ATG9 participates in an early step by shuttling from a non-PAS site to the PAS site and supports an assembly model for yeast autophagosome biogenesis (4). In contrast, mammalian ATG9 is not stably incorporated into the isolation membrane or autophagosomes but is instead transiently associated with the omegasome, a phosphatidylinositol 3-phosphate (PI3P)-enriched endoplasmic reticulum (ER) subdomain (5). Cryomicroscopy studies have shown a close association between ATG9 vesicles and the omegasome structure (7), together with the presence of ATG9 on tubulovesicular membranes surrounding autophagosomes (5). A recent finding by livecell imaging indicates that autophagosome formation occurs where ATG9 vesicles coalesce with the ER ...

Eukaryotic cells use autophagy to recycle cellular components. During autophagy, autophagosomes deliver cytoplasmic contents to the vacuole or lysosome for breakdown. Mammalian cells regulate the dynamics of autophagy via ubiquitinmediated proteolysis of autophagy proteins. Here, we show that the Arabidopsis thaliana Tumor necrosis factor ReceptorAssociated Factor (TRAF) family proteins TRAF1a and TRAF1b (previously named MUSE14 and MUSE13, respectively) help regulate autophagy via ubiquitination. Upon starvation, cytoplasmic TRAF1a and TRAF1b translocated to autophagosomes. Knockout traf1a/b lines showed reduced tolerance to nutrient deficiency, increased salicylic acid and reactive oxygen species levels, and constitutive cell death in rosettes, resembling the phenotypes of autophagy-defective mutants. Starvation-activated autophagosome accumulation decreased in traf1a/b root cells, indicating that TRAF1a and TRAF1b function redundantly in regulating autophagosome formation. TRAF1a and TRAF1b interacted in planta with ATG6 and the RING finger E3 ligases SINAT1, SINAT2, and SINAT6 (with a truncated RING-finger domain). SINAT1 and SINAT2 require the presence of TRAF1a and TRAF1b to ubiquitinate and destabilize AUTOPHAGY PROTEIN6 (ATG6) in vivo. Conversely, starvation-induced SINAT6 reduced SINAT1-and SINAT2-mediated ubiquitination and degradation of ATG6. Consistently, SINAT1/SINAT2 and SINAT6 knockout mutants exhibited increased tolerance and sensitivity, respectively, to nutrient starvation. Therefore, TRAF1a and TRAF1b function as molecular adaptors that help regulate autophagy by modulating ATG6 stability in Arabidopsis.