Autophagosomes

by Arik Honig & Tamar Avin-Wittenberg

Department of Plant Sciences. The Weizmann Institute of Science, Israel.

Macroautophagy (hereafter referred to as “Autophagy”) is a conserved eukaryotic mechanism, which is classically defined as the degradation of cytoplasmic constituents in the lytic organelle (vacuoles in yeast and plants and lysosomes in mammals) [1]. The general targets of autophagy vary from long-lived proteins to protein complexes and even organelles, and it can be either bulk or selective [2]. Morphologically, autophagy begins in the formation of cup-shaped double membranes, which expand to form autophagosomes engulfing malfunctioning or un-needed macromolecules and organelles and transport them for degradation inside the vacuole. Upon arrival of the autophagosomes to the vacuoles, their outer membrane fuses with the tonoplast, creating single membrane vesicles inside the vacuole, termed 'autophagic bodies'. The autophagic bodies and their contents are then degraded inside the vacuole, providing recycled materials to build new macromolecules [3]. The genes participating in the autophagic process (termed AuTophaGy-related or ATG genes) were originally discovered in yeast (Saccharomyces cerevisiae), using autophagy-defective mutants whose cells show little or no accumulation of autophagic bodies during nutrient starvation [1].

Many of the ATG genes are conserved in evolution and homologs of the yeast genes have been found in many organisms, including mammals and plants [2,4,5]. The core autophagy machinery is divided into three groups (i) the ATG9 cycling system, including ATG1, ATG2, ATG9, ATG13, ATG18 and ATG27; (ii) the PI3-kinase complex, comprised of ATG6/VPS30/BECLIN1, ATG14, VPS15 and VPS34; and (iii) the ubiquitin-like protein system, comprised of ATG3, ATG4, ATG5, ATG7, ATG8, ATG10, ATG12 and ATG16 [for examples, see the following reviews: [1,4,5]. In the past few years, our knowledge of the functions of autophagy in plants has been greatly expanded. Autophagy in plants has been shown to occur at basal levels under favorable (non-stress) growth conditions [6,7] and was also shown to be involved in response to various abiotic and biotic stresses as well as in hormonal regulation [ 8-12]. It is also important to note that degradation of the major plastid protein RUBISCO as well as entire chloroplasts during senescence was shown to be performed by autophagy [13,14]. Another fascinating aspect is the emerging role of autophagy in plant-pathogen interactions [11,15]. Most of these roles were discovered using various Arabidopsis thaliana T-DNA knock-out mutants, but some were also identified with the help of fluorescent markers (see Table). The markers used for autophagy research in plants are based almost completeley on the ATG8 isoforms. Arabidopsis possesses nine genes encoding ATG8 isoforms. The ATG8‘s were divided into three sub-families according to protein sequence similarity: AtATG8a, AtATG8c, AtATG8d and AtATG8f (4 members), AtATG8b, AtATG8e and AtATG8g (3 members) and AtATG8h and AtATG8i (2 members) [16]. Mechanistically, ATG8 is one of two proteins containing a ubiquitin-fold in the autophagy core machinery. It is synthesized as a pro-protein that is cleaved by ATG4 to expose a glycine at the C-terminus of the protein (This is also the reason it can be tagged with a fluorescent marker only on its N-terminus). In a ubiquitin-like conjugation system, the processed ATG8 protein then binds through the exposed glycine to phosphatidylethanolamine (PE) molecules on membranes that are programmed to differentiate into autophagosomes [4]. ATG8-PE located on the outer membrane of the autophagosome is cleaved off during autophagosome deposition by ATG4. ATG8-PE is anchored to the inner membrane of the autophagosome, which moves in the cytosol and eventually enters the vacuole where the autophagic body is degraded [17]. This cellular route makes the ATG8 protein an excellent marker to follow autophagosomes during autophagy.

Many groups have independently constructed fluorescent GFP-ATG8 reporters that are widely used in plant autophagy research [1,9,18,19, 20]. Moreover, with the assistance of ConcanamycinA it is also possible to visualize the GFP-ATG8 protein inside the vacuole [21]; (Accompanying figure shows vacuole containing autophagic bodies in a hypocotyl cell of a carbon starved seedling of GFP-ATG8f treated with ConA ). Two other Arabidopsis ATG proteins, ATG12 and ATG18 were GFP tagged recently [17, 22], but unfortunatley, the fluorescence pattern of the chimeric proteins was cytosolic or nuclear, limiting their use for autophagosome tracking. Remarkebly, the current autophagy cellular markers are limited just for the Arabidopsis proteins described above.

Furthermore, ATG8 in yeast and mammals has been shown to mediate target recognition during selective autophagy by specific binding to the target sequence AIM (ATG8 Interacting motif; [23]). AIMs are evolutionary conserved among the large family of ATG8-binding proteins in various species [23]. Despite the extensive identification of AIM-containing ATG8-binding proteins in mammals and yeast, there are so far only three recent publications on AIM-containing ATG8-binding proteins in plants [12, 24, 25 ]. An Arabidopsis AtNBR1 homolog has been identified as a selective autophagy substrate [24]. The tobacco NtJoka2 gene was identified as the homolog of AtNBR1 [25] and it has been shown that the Arabidopsis TSPO, a sensory protein localized to the cell plasma membrane, possesses an AIM and is degraded through selective autophagy and not through the proteasome pathway [12]. In all three cases, the selective autophagy targets were also labled with fluorescent tags and were shown to co-localize with ATG8 in-vivo. Thus, these three newly discovered ATG8 binding proteins are the first plant protein markers for selective autophagy processes, and apparently will contribute greatly to the cellular research in this field.

List of fluorescent protein probes for autophagosomes in Arabidopsis thaliana.

References

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2. Reumann S, Voitsekhovskaja O, Lillo C (2010) From signal transduction to autophagy of plant cell organelles: lessons from yeast and mammals and plant-specific features. Protoplasma 247(3-4): 233-256

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12. Vanhee C, Zapotoczny G, Masquelier D, Ghislain M, Batoko H (2011) The Arabidopsis Multistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism. Plant Cell 23(2): 785-805

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23. Noda NN, Ohsumi Y, Inagaki F (2010) Atg8-family interacting motif crucial for selective autophagy. FEBS Lett 584(7): 1379-1385

24. Svenning S, Lamark T, Krause K, Johansen T (2011) Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1. Autophagy 7 (9): 1-18

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26. Suttangkakul A, Li F, Chung T, Vierstra RD (2011) The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis. Plant Cell. Oct 7 [Epub ahead of print].

External Links :Autophagy. Plant Organellome database .