LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis

Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE‐16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE‐16 subfamily is essential for a later stage in autophagosome maturation.     

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Essential and distinct roles of phosphatidylinositol 4-kinases,Pik1p and Stt4p,in yeast autophagy

Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome–vacuole fusion in yeast cells, respectively.     

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B,GABARAPL2 and GABARAP

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.     

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1? and sjl3?, and the temperature-sensitive mutant sac1^ts/sjl3? at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1? and sac1^ts/sjl3? mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1? strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1? strain, although autophagosomes were present in the cytoplasm. In the sac1^ts/sjl3? strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3? strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.     

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.     

Curvature‐sensitive trans‐assembly of human Atg8‐family proteins in autophagy‐related membrane tethering

In macroautophagy, de novo formation of the double membrane‐bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8‐family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8‐mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE‐16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE‐16 retained the capacities to trigger efficient membrane tethering at the protein‐to‐lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8‐mediated membrane‐tethering reactions require trans‐assembly between the membrane‐anchored forms of LC3B and GATE‐16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B‐ and GATE‐16‐mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE‐16 for highly curved small vesicles (e.g., 50 nm in diameter), although GATE‐16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g., 200 and 400 nm in diameter). Our findings establish curvature‐sensitive trans‐assembly of human Atg8‐family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.     

Mammalian Atg8s: One is simply not enough

Yeast Atg8, a key factor in the autophagic process, is a ubiquitin-like protein that undergoes a unique conjugation to phosphatidylethanolamine (PE). Atg8 plays a dual role in early stages of autophagosome formation: It was implicated in recruitment of cargo proteins such as Atg19 and Atg32 for Cvt and mitophagy, respectively, and in autophagosome biogenesis, serving as an elongation factor by mediating membrane hemi-fusion. Similarly, the mammalian Atg8 proteins, LC3s and GABARAPs, recruit cargo into autophagosomes by binding to adaptor proteins such as p62, NBR1 and Nix. These functions, however, are not essential for bulk autophagic flux. Other studies in which the activity of the mammalian Atg8s was blocked either by knockout of the E2-like enzyme Atg3 or by using a dominant negative mutant of the promiscuous protease Atg4B revealed, in agreement with the yeast Atg8 data, that the mammalian factors are crucial for the formation of normal and mature autophagosomes. While it seems that the single yeast Atg8 and the mammalian Atg8s share similar roles, it is still unclear why the mammalian system employs several homologs. Recent publications demonstrated that the mammalian Atg8s differ in their cargo specificity, as Nix, for example, binds exclusively to GABARAP-L1. This may suggest that these proteins exhibit distinct activity also in autophagosome biogenesis. In our study we divided the mammalian Atg8s into two subfamilies of homologs based on amino acid similarity, the LC3 and GABARAP/GATE-16 subfamilies, and tested their essentiality and role in autophagy. In agreement with previous studies we found that the mammalian Atg8s are essential for autophagy but, more importantly, that each of these subfamilies has a distinct role in the process of autophagosome biogenesis.     

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum‐derived vesicles to Legionella‐containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4‐phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P‐binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.     

Dynamics and function of PtdIns(3)P in autophagy

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 3-kinase is essential for autophagy. However, the distribution and function of the enzymatic product, PtdIns 3-phosphate (PtdIns(3)P), has been unknown. We monitored PtdIns(3)P distribution during autophagy by live imaging, biochemistry, and electron microscopy, and found that PtdIns(3)P is massively delivered into the vacuole via autophagy. PtdIns(3)P is highly enriched as a membrane component of the elongating isolation membranes and autophagosome membranes rather than as an enclosed cargo, implying direct involvement of PtdIns(3)P in autophagosome formation. This observation also provides important basic information on the nature of the autophagosome membrane, which is still poorly understood. Notably, PtdIns(3)P is highly enriched on the inner (concave) surfaces of the isolation membrane and autophagosome compared to the outer surfaces. PtdIns(3)P is also enriched on ambiguous structures juxtaposed to the elongating tips of isolation membranes. We also investigated the function of PtdIns(3)P in autophagy, and show that PtdIns(3)P recruits the Atg18-Atg2 complex to autophagic membranes through an Atg18-PtdIns(3)P interaction. Interestingly, PtdIns(3)P is required only for the association of the Atg18-Atg2 complex to autophagic membranes but not for any subsequent functional activity of the Atg18-Atg2 complex, suggesting that PtdIns(3)P does not act allosterically on Atg18. Based on these results we discuss the function of PtdIns(3)P in autophagy.     

GABARAPL1 (GEC1) associates with autophagic vesicles

Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.     

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Phosphatidylinositol 3,5‐Bisphosphate‐Rich Membrane Domains in Endosomes and Lysosomes

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P₂) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P₂ using freeze‐fracture electron microscopy. GST‐ATG18‐4×FLAG was used to label PtdIns(3,5)P₂ and its binding to phosphatidylinositol 3‐phosphate (PtdIns(3)P) was blocked by an excess of the p40^phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P₂ was concentrated in intramembrane particle (IMP)‐deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP‐deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid‐disordered domain marker. In yeast lacking either PtdIns(3,5)P₂ or its effector, Atg18p, the IMP‐deficient, PtdIns(3)P‐rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P₂ metabolism is defective. In HeLa cells, PtdIns(3,5)P₂ was significantly enriched in the vesicular domain of RAB5‐ and RAB7‐positive endosome/lysosomes of the tubulo‐vesicular morphology. This biased distribution of PtdIns(3,5)P₂ was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P₂‐rich and ‐deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.      

GABARAPL1 antibodies: Target one protein,get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA_A receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Direct link between Atg protein and small GTPase Rab: Atg16L functions as a potential Rab33 effector in mammals

Atg16L is a factor that is essential for elongation of the isolation membrane (also called phagophore), a precursor of the autophagosome. Atg16L facilitates LC3/Atg8-conjugation to phosphatidylethanolamine by forming an oligomeric complex with Atg12-conjugated Atg5 and recruiting an LC3-Atg3 intermediate to elongating isolation membranes. Although Atg16L is responsible for the isolation membrane localization of the complex, the mechanism by which Atg16L is targeted to or recognizes isolation membranes remains largely unknown. We recently reported finding that Atg16L specifically and directly interacts with the Golgi-resident small GTPase Rab33B (and Rab33A) via the coiled-coil domain of Atg16L. Since expression of a GTPase-deficient mutant of Rab33B or the coiled-coil domain of Atg16L modulates macroautophagy (simply referred to as autophagy below), Atg16L (or the Atg12-5/16L complex) is likely to function as a specific effector molecule for Rab33 in autophagosome formation. Future study of the cross talk between Atg16L-mediated autophagosome formation and Rab33-mediated membrane trafficking should provide an important clue to unresolved issues in autophagosome formation, specifically, the membrane source of autophagosomes.     

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100?nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.