Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogenlimiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.Protein turnover plays numerous essential roles in plants, including the precise removal of short-lived regulatory proteins, the elimination of abnormal proteins, the maintenance of amino acid pools needed for continual protein synthesis, and the recycling of carbon (C) and nitrogen (N) during senescence and apoptosis (Vierstra, 1996). Whereas much of regulatory protein turnover is accomplished by the highly selective ubiquitin (Ub)/26S proteasome pathway (Smalle and Vierstra, 2004), plants, like other eukaryotes, also use less-selective high-throughput mechanisms for degrading intracellular constituents in bulk. One of the most conspicuous in the cytoplasm is autophagy. Here, individual proteins, protein complexes, and entire organelles become engulfed in membrane-bound vesicles, which are delivered to the vacuole (yeast/plants) or lysosome (animals; Ohsumi, 2001;Klionsky, 2004;Thompson and Vierstra, 2005). These vesicles and their contents are then degraded by a wide range of vacuolar proteases, peptidases, lipases, and other hydrolytic enzymes.In yeasts, autophagic engulfment and delivery to the vacuole occurs by two morphologically distinct but mechanistically overlapping routes, microautophagy and macroautophagy (Klionsky, 2004). Microautophagy involves the sequestration of cytosol by tubular invaginations of the tonoplast. These tubes then pinch off to release intravacuolar vesicles ca...

Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG1a and ATG13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant's nutritional status.

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