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.
autophagosome-gfp
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. 
Probes for autophagosomes

References

1. Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9(10): 1102-1109

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

3. Bassham DC (2009) Function and regulation of macroautophagy in plants. Biochim Biophys Acta 1793(9): 1397-1403

4. Tanida I (2011) Autophagosome Formation and Molecular Mechanism of Autophagy. Antioxid Redox Signal 14(11): 2201-2214

5. Avin-Wittenberg T, Honig A, Galili G (2011) Variations on a theme: plant autophagy in comparison to yeast and mammals. Protoplasma (in press).

6. Sláviková S, Shy G, Yao Y, Glozman R, Levanony H, Pietrokovski S, Elazar Z, Galili G (2005) The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants. J Exp Bot 56(421): 2839-2849

7. Inoue Y, Suzuki T, Hattori M, Yoshimoto K, Ohsumi Y, Moriyasu Y (2006) AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells. Plant Cell Physiol 47(12): 1641-1652

8. Xiong Y, Contento AL, Nguyen PQ, Bassham DC (2007) Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol 143(1): 291-299

9. Slavikova S, Ufaz S, Avin-Wittenberg T, Levanony H, Galili G (2008) An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses. J Exp Bot 59(14): 4029-4043

10. Liu Y, Xiong Y, Bassham DC (2009) Autophagy is required for tolerance of drought and salt stress in plants. Autophagy 5(7): 954-963

11. Hayward AP, Dinesh-Kumar SP (2010) What can Plant Autophagy Do for an Innate Immune Response? Annu Rev Phytopathol 49: 4.1-4.20

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

13. Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T (2008) Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiol 148(1): 142-155

14. Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A (2009) Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol 149(2): 885-893

15. Cacas JL (2010) Devil inside: does plant programmed cell death involve the endomembrane system? Plant Cell Environ 33(9): 1453-1473

16. Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD (2002) The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem 277(36): 33105-33114

17. Chung T, Phillips AR, Vierstra RD (2010) ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J 62(3): 483-493

18. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 16(11): 2967-2983

19. Contento AL, Xiong Y, Bassham DC (2005) Visualization of autophagy in Arabidopsis using the fluorescent dye monodan-sylcadaverine and GFP-AtATG8e fusion protein. Plant J 42 (4): 598-608. 

20. Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 138(4): 2097-2110

21. Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A, Higashi SI, Watanabe M, Nishimura M, Hara-Nishimura I (2003). Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J. 35, 545-555.

22. Lai Z, Wang F, Zheng Z, Fan B, Chen Z (2011) A critical role of autophagy in plant resistance to necrotrophic fungal pathogens. Plant J 66: 953-968

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

25. Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M, Liszewska F, Wawrzynska A, Sirko A (2011) Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors. Autophagy 7 (10): 1145-1158.

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 .