9.A.15 The Autophagy-related Phagophore-formation Transporter (APT) Family
Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and it plays a key role in membrane transport as a phospholipid scramblase (Nakao and Nakano 2022); the phagophore presumably expands by vesicular addition to form a complete autophagosome. Atg9 functions at the phagophore assembly site (PAS) (Munakata and Klionsky, 2010). He et al. (2008) reported that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses revealed that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation. It includes 6 putative TMSs.
Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins (Umemiya et al., 2007). Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, Atg, have been identified that are required for autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell (Xie and Klionsky, 2007).
In yeast, approximately 31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS). Geng et al. (2008) reported the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. They found that an increase in the amount of Atg11 at the PAS enhanced the recruitment of Atg8 and Atg9 to this site and facilitated the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remained unchanged at the PAS, whereas an enhanced recruitment of Atg8 and 9 at this site was observed. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes, and both Atg8 and Atg12 were ubiquitinylated (Geng and Klionsky, 2008). Novel Atg proteins and the stages of their action have been identified (Backues et al. 2015).
Atg9 is the only characterized transmembrane protein that is absolutely required for Cvt vesicle formation, and it is proposed to carry membrane from peripheral donor sites to the phagophore assembly site where the vesicle forms. It is also a lipid scramblase that mediates autophagosomal membrane expansion (Matoba et al. 2020). Additional proteins, including Atg11, Atg23, and Atg27, are involved in the anterograde movement, whereas Atg1-Atg13 and Atg2-Atg18 are required for the retrograde return to the peripheral sites (Munakata and Klionsky, 2010). Atg11 and Atg23 show low sequence similarity to MLP1 and MLP2 of the Nuclear Pore Complex (NPC; 1.A.75.1.1). These proteins include repeat sequences. Atg1 is a serine, thereonine protein kinase; also called autophagy protein 3 or cytoplasm to vacuole targeting protein 10.
Intestinal Paneth cells limit bacterial invasion by secreting antimicrobial proteins, including lysozyme. However, invasive pathogens can disrupt the Golgi apparatus, interfering with secretion and compromising intestinal antimicrobial defense. Bel et al. 2017 showed that during bacterial infection, lysozyme is rerouted via secretory autophagy, an autophagy-based alternative secretion pathway. Secretory autophagy was triggered in Paneth cells by bacteria-induced endoplasmic reticulum (ER) stress, required extrinsic signals from innate lymphoid cells, and limited bacterial dissemination. Secretory autophagy was disrupted in Paneth cells of mice harboring a mutation in autophagy gene Atg16L1 that confers increased risk for Crohn's disease in humans. These findings identify a role for secretory autophagy in intestinal defense and suggest why Crohn's disease is associated with genetic mutations that affect both the ER stress response and autophagy (Bel et al. 2017).
The lysosome (or vacuole in yeast) is the central organelle responsible for cellular degradation and nutrient storage. Lysosomes receive cargo from the secretory, endocytic, and autophagy pathways. Many of these proteins and lipids are delivered to the lysosome membrane, and some are degraded in the lysosome lumen, whereas others appear to be recycled. Suzuki and Emr 2018 identified the transmembrane autophagy protein Atg27 as a physiological cargo recycled from the vacuole. Atg27 is delivered to the vacuole membrane and then recycled using a two-step recycling process. First, Atg27 is recycled from the vacuole to the endosome via the Snx4 complex and then from the endosome to the Golgi via the retromer complex TC# 9.A.3). During the process of vacuole-to-endosome retrograde trafficking, Snx4 complexes assemble on the vacuolar surface and recognize specific residues in the cytoplasmic tail of Atg27. This pathway maintains the normal composition and function of the vacuole membrane (Suzuki and Emr 2018).
The table (Table 3) presented below is taken from (Munakata and Klionsky 2010).
Autophagy-related protein complex of Saccharomyces cerevisiae (Munakata and Klionsky, 2010). Different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Phosphorylation regulates the Atg9 interaction with Atg23 and Atg27 (Feng et al. 2016). Atg27 is required for Atg9 cycling, and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex (Yen et al. 2007). Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Matoba et al. 2020 reported that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 revealed a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. Thus, phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion. Guardia et al. 2020 solved a high-resolution cryoEM structure of the ubiquitously expressed human ATG9A isoform. ATG9A is a domain-swapped homotrimer with a unique fold, and has an internal network of branched cavities. The functional importance of the cavity-lining residues which could serve as conduits for transport of hydrophilic moieties, such as lipid headgroups, across the bilayer has been suggested (Guardia et al. 2020). Transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes (Orii et al. 2021).
Autophagy-related protein complex of Saccharomyces cerevisiae
Atg9 (997 aas; 6TMSs) (Q12142) (homologous to Q5ANC9 below)
Atg11 (1178 aas; 0TMSs) (Q12527)
Atg23 (453 aas; 0 TMSs) (Q06671)
Atg27 (271 aas; ≤ 2 TMSs) (P46987)
Atg1 (897 aas; ≤ 2 TMSs) (P53104)
Atg2 (1592 aas; ≤ 2 TMSs) (P53855)
Atg13 (738 aas; ≤ 2 TMSs) (Q06628)
Atg18 (500 aas; ≤ 2 TMSs) (P43601)
Atg9 (528 aas) (Q5ANC9) (homologous to Q12142 above)
Atg22 (528 aas; 12 TMSs) (P25568) This protein is a member of the MFS and also has TC# 2.A.1.24.1.
The autophagy protein complex. The molecular mechanisms of autophagy have been reviewed (Hurley and Young 2017; Dupont et al. 2017). Autophagy is related to apoptosis and autoimmunity (Song et al. 2017; Wu and Adamopoulos 2017). It is an intracellular degradation process carried out by a double-membrane organelle, termed the autophagosome (Molino et al. 2017). Three proteins (TM9SF1 (TC#8.A.68.1.13), TMEM166 (listed here) and TMEM74 (TC# 9.B.189.2.1)) regulate autophagosome formation (He et al. 2009). The generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy. ATG9A is essential for multiple steps of epithelial tight junction biogenesis and actin cytoskeletal regulation (Dowdell et al. 2020). Autophagy involves capture of cytoplasmic materials into double-membraned autophagosomes that subsequently fuse with lysosomes for degradation of the materials by lysosomal hydrolases. The cryoelectron microscopy structure of the human ATG9A isoform at 2.9-Å resolution has been solved (Guardia et al. 2020). The structure reveals a fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a membrane transporter. Mutational analyses support a role for the cavities in the function of ATG9A. Structure-guided molecular simulations predict that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes (Guardia et al. 2020). The mechanism of Atg9 recruitment by Atg11 in the cytoplasm-to-vacuole targeting pathway has been examined (Coudevylle et al. 2022). Autophagosomes form de novo, but how is poorly understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. Sawa-Makarska et al. 2020 reconstituted autophagosome nucleation using recombinant components from yeast. They found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. They found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to cargo via Atg19-Atg11-Atg9 interactions. They proposed that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum (Sawa-Makarska et al. 2020). Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena (Kiss et al. 2020). RUSC2 and WDR47 oppositely regulate kinesin-1-dependent distribution of ATG9A to the cell periphery (Guardia et al. 2021). The adaptor protein chaperone AAGAB (TC family 8.A.203) stabilizes AP-4 complex subunits (Mattera et al. 2022).
The autophagy protein complex of Homo sapiens
ATG14 of 492 aas (Q6ZNE5)
ATG7 of 703 aas (O95352)
ATG4B of 393 aas (Q9Y4P1)
ATG9A of 839 aas and 6 - 8 TMSs (Q7Z3C6)
ATG16L1 of 607 aas (Q676U5)
ATG13 of 517 aas (O75143)
ATG4D of 474 aas (Q86TL0)
ATG3 of 314 aas (Q9NT62)
ATG12 of 140 aas (O94817)
ATG101 of 218 aas (Q9BSB4)
ATG2 of 1,938 aas (Q2TAZ0)
ATG9B of 924 aas (Q674R7)
ATG2B of 2078 aas (Q96BY7)
ATG16L2 of 619 aas (Q8NAA4)
ATG5 of 275 aas (A9UGY9)
Plant autophagy protein complex. This complex plays roles in development and stress responses (Lv et al. 2014). Autophagic regulators and receptors have been identified (Zeng et al. 2016). Autophagy may play a role in sexual reproduction (Kurusu and Kuchitsu 2017). LptB2FG displays adenylate kinase activity in vitro dependent on binding partners LptC/LptA (Baeta et al. 2021). Cyclopaldic acid, the main phytotoxic metabolite of Diplodia cupressi, induces programmed cell death and autophagy in Arabidopsis thaliana (Samperna et al. 2022).
Autophagy complex of Arabidopsis thaliana (Mouse-ear cress)
ATG18A of 425 aas (Q93VB2)
ATG6 of 517 aas (Q9M367)
ATG8A of 137 aas (A8MS84)
ATG7 of 697 aas (Q94CD5)
ATG11 of 1,148 aas (Q9SUG7)
ATG10 of 225 aas (Q8VZ52)
ATG1A of 626 aas (Q94C95)
ATG5 of 337 aas (Q9FFI2)
ATG4B of 477 aas (Q9M1Y0)
ATG9 of 866 aas (Q8RUS5)
ATG3 of 313 aas (Q0WWQ1)
ATG13A of 603 aas (Q9SCK0)
ATG2 of 1,892 aas (F8S296)
ATG12A of 96 aas (Q8S924)
ATG1C of 733 aas (F4IRW0)