GABARAP
Introduction
GABARAP - Gamma-Aminobutyric Acid Receptor-Associated Protein.
Lipidation of GABARAP
GABARAP lipidation is a critical post-translational modification essential for autophagy, a cellular degradation pathway that delivers cytoplasmic components to lysosomes.
This process involves the conjugation of GABARAP to phosphatidylethanolamine (PE) in autophagosomal membranes, enabling GABARAP to function in cargo recruitment and membrane biogenesis.
The lipidation of GABARAP follows a ubiquitin-like conjugation system that requires multiple enzymes working in sequence.
The final step in the sequence is transfer of GABARAP/LC3 (a family of proteins collectively called ATG8) from ATG3 to phosphatidylethanolamine (PE) in the membrane (R1).
ATG8 is covalently linked to ATG3 by an activation mechanism involving the ATPase ATG7 (that is, ATP is consumed?).
ATG3–ATG8 then adheres to and partly inserts into the membrane through the action of an N-terminal amphipathic helix in ATG3, which orients the activated ATG8 in correct position for the transfer to the PE substrate. (R1)
Lipidation of GABARAP is not attaching a lipid to the protein, it’s the opposite - it’s linking the GABARAP-ATG3 complex to the membrane to PE part of the phospholipid layer.
This delivery and insertion appears to be facilitated by the complex ATG12-ATG5-ATG16L1 (R1).
Step 1: Priming of GABARAP by ATG4 Proteases
ATG4 Proteases and Their Function
The first step in GABARAP lipidation is the proteolytic processing of newly synthesized pro-GABARAP by ATG4 cysteine proteases.
This priming step is essential as it exposes the C-terminal glycine residue (G116) of GABARAP, which is required for subsequent conjugation reactions.
In mammals, four ATG4 isoforms (ATG4A, ATG4B, ATG4C, and ATG4D) have been identified, with ATG4B showing the highest activity toward GABARAP and its family members.
ATG4B contains a catalytic cysteine residue (Cys74) that is essential for its proteolytic activity.
Mechanism of ATG4-Mediated Priming
The priming reaction involves the cleavage of pro-GABARAP after the C-terminal glycine residue, removing any amino acids that follow it. This cleavage is facilitated by the interaction between GABARAP and ATG4B, where a conserved arginine residue (Arg68) in GABARAP forms a salt bridge with Asp171 of ATG4B.
This interaction is critical for proper positioning of the substrate for cleavage, and mutations affecting this salt bridge significantly reduce ATG4B’s ability to process GABARAP.
Step 2: Activation by ATG7 (E1-like Enzyme)
ATG7 Structure and Function
Following priming, the exposed C-terminal glycine of GABARAP is activated by ATG7, which functions as an E1-like activating enzyme in this ubiquitin-like conjugation system. ATG7 is a homodimeric protein that binds ATP and catalyzes the adenylation of GABARAP’s C-terminal glycine.
Mechanism of ATG7-Mediated Activation
The activation process begins with the binding of ATP and GABARAP to ATG7. ATG7 then catalyzes the formation of a high-energy GABARAP-adenylate intermediate, with the release of pyrophosphate.
This adenylation reaction requires ATP as a cofactor and results in the formation of a thioester bond between the C-terminal glycine of GABARAP and a catalytic cysteine residue in ATG7.
This GABARAP~ATG7 thioester intermediate is then ready for transfer to the E2-like enzyme ATG3.
Step 3: Transfer to ATG3 (E2-like Enzyme) and Conjugation to PE
ATG3 Structure and Function
ATG3 serves as the E2-like conjugating enzyme in GABARAP lipidation. It receives the activated GABARAP from ATG7 through a transthioesterification reaction, forming a GABARAP~ATG3 thioester intermediate. ATG3 contains a catalytic cysteine residue that forms this thioester bond with GABARAP’s C-terminal glycine.
Membrane Curvature Sensing by ATG3
A unique feature of ATG3 is its ability to sense membrane curvature through an N-terminal amphipathic helix. This helix preferentially binds to membranes with lipid-packing defects, which are typically found at highly curved regions such as the edge of the growing phagophore. This property ensures that GABARAP lipidation occurs preferentially at the appropriate membrane sites.
GABARAP~ATG3 Thioester Intermediate
Recent structural studies have provided insights into the GABARAPATG3 conjugate. The crystal structure reveals that this conjugate adopts an open configuration with minimal contacts between the two proteins. Interestingly, non-covalent contacts between GABARAP and the backside of ATG3’s catalytic center have been observed, forming a helical filament of the GABARAPATG3 conjugate. This non-covalent interaction plays a critical role in the PE conjugation process.
Step 4: E3-like Complex and Final Conjugation to PE
ATG12–ATG5-ATG16L1 Complex
The final step in GABARAP lipidation is facilitated by the ATG12–ATG5-ATG16L1 complex, which functions as an E3-like ligase. This complex enhances the efficiency of GABARAP conjugation to PE by bringing the GABARAP~ATG3 thioester intermediate into proximity with the target membrane.
The ATG12–ATG5-ATG16L1 complex is formed through another ubiquitin-like conjugation system, where ATG12 is covalently attached to ATG5, and this conjugate then non-covalently binds to ATG16L1. The resulting complex has multiple membrane-binding regions in ATG16L1 that contribute to its localization to autophagosomal membranes.
Three-Step Docking Mechanism
Recent research has revealed a three-step docking mechanism by which the E3-like complex delivers GABARAP to the phagophore membrane: 1. Initial recruitment by the phosphatidylinositol 3-phosphate (PI3P) effector protein WIPI2 2. Membrane interaction through helix α2 of ATG16L1 3. Final positioning by a membrane-interacting surface of ATG3
This coordinated process ensures precise targeting of GABARAP to the appropriate membrane sites.
Final Conjugation to PE
The final step involves the transfer of GABARAP from the GABARAP~ATG3 thioester intermediate to the amino group of PE in the target membrane.
This reaction results in the formation of an amide bond between GABARAP’s C-terminal glycine and the amino group of PE. The presence of the ATG12–ATG5-ATG16L1 complex significantly increases the rate and efficiency of this conjugation reaction.
Interestingly, recent studies have shown that PE lipids concentrate in the region around the thioester bond between ATG3 and GABARAP, with two conserved histidine residues in ATG3 potentially playing a role in the catalytic transfer of GABARAP to PE.
Regulation of GABARAP Lipidation
ATG4-Mediated Delipidation
In addition to their role in priming, ATG4 proteases also function in delipidation, the process of removing GABARAP from membranes.
This deconjugation activity is important for maintaining the proper balance of lipidated and non-lipidated GABARAP pools.
Recent studies suggest that ATG4D may play a particularly important role in the delipidation of GABARAP from autophagosomal and autolysosomal membranes.
Note: ATG4 proteases are sensitive to H2O2:
We show that starvation stimulates formation of ROS, specifically H(2)O(2).
These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins.
Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H(2)O(2), and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation.(R4)
E3-like Complex Regulation
The ATG12–ATG5-ATG16L1 complex not only enhances GABARAP lipidation but also regulates the subsequent functions of lipidated GABARAP. While this complex increases and accelerates GABARAP lipidation and vesicle tethering, it can also hamper GABARAP’s capacity to induce inter-vesicular lipid mixing or fusion, possibly through the formation of a rigid scaffold on the vesicle surface.
Differences Between GABARAP and LC3 Lipidation
While the lipidation machinery is shared between GABARAP and LC3 subfamilies, there are notable differences in their lipidation efficiency and subsequent functions. In the absence of the E3-like complex, GABARAP and GABARAPL1 show higher lipidation and membrane tethering activities compared to LC3 proteins. This suggests a lower lipidation threshold for the GABARAP subfamily as a prerequisite for tethering and inter-vesicular lipid mixing.
ATG16L1
Plays an essential role in both canonical and non-canonical autophagy: interacts with ATG12-ATG5 to mediate the lipidation to ATG8 family proteins (MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAPL1, GABARAPL2 and GABARAP) (Uniprot)
Under starved conditions, the ATG12-ATG5-ATG16L1 complex is translocated to phagophores driven by RAB33B (R2)
ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation.
Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. (R2)
GABARAPs are a positive regulator of ULK1
By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity.
To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing.
The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. (R3)