Academic journal article Genetics

The ATG12-Conjugating Enzyme ATG10 Is Essential for Autophagic Vesicle Formation in Arabidopsis Thaliana

Academic journal article Genetics

The ATG12-Conjugating Enzyme ATG10 Is Essential for Autophagic Vesicle Formation in Arabidopsis Thaliana

Article excerpt

ABSTRACT

Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants.

AS with other eukaryotes, plants have developed sophisticated mechanisms to recycle intracellular proteins. Most selective protein turnover occurs by the ubiquitin (Ub)/26S proteasome pathway, which directs the correct removal of short-lived regulatory and abnormal proteins (Smalle and Vierstra 2004). Conversely, autophagy is a catabolic process that is largely responsible for nonselective bulk turnover of cytosolic components from individual proteins and protein complexes to the removal of whole organelles (Thompson and Vierstra 2005; Bassham 2007). It involves the engulfment of cytoplasm in small vesicles followed by their deposition into the lytic vacuole (lysosome in animals) where the vesicles and cargo are quickly degraded by a cache of vacuolar proteases, peptidases, lipases, and other hydrolytic enzymes.

Thus far, primarily using the yeasts Saccharomyces cerevisiae and Pichia pastoris as models, at least two autophagic routes have been identified (for reviews see Ohsumi 2001; Thompsonand Vierstra 2005; Klionsky 2007). Microautophagy proceeds by forming tubular invaginations of cytoplasm into the vacuole, which pinch off and release vesicles called autophagic bodies into the vacuolar lumen. In contrast, macroautophagy involves the de novo formation of small double-membrane-bound vesicles called autophagosomes within the cytoplasm, which sequester cytosolic constituents. These vesicles dock with the vacuole, where the outer membrane fuses with the tonoplast to release the inner compartment into the vacuolar lumen as an autophagic body. In addition, a derivative of macroautophagy called the cytoplasm-to-vacuole targeting (CVT) pathway exists to encapsulate and deliver functional proteins such as preaminopeptidase to the vacuole (Klionsky 2007). While the CVT pathway has been confirmed only in S. cerevisiae, it is possible that a similar vacuolar transport pathway is active in plants (Thompson and Vierstra 2005; Seay et al. 2006). Bothmicro- and macroautophagy are essential in yeast for maintaining nitrogen (N) and carbon (C) pools, recycling amino acids, removing unwanted or damaged organelles, and survival during starvation. Additional roles in programmed cell death (PCD) and various pathologies have been observed in animals (Bursch 2001; Levine and Klionsky 2004; Ueno et al. 2004; Juhasz et al. 2007).

Through genetic dissection of autophagy in yeasts over the past decade, several groups have discovered a set of autophagy (ATG) proteins common to both micro- and macroautophagy (Tsukada and Ohsumi 1993; Thumm et al. …

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