Class III phosphatidylinositol 3-kinase complex I subunit NRBF2/Atg38 - from cell and structural biology to health and disease

Abstract

Macroautophagy/autophagy is triggered by various starvation and stress conditions. The phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) is essential for the formation of the autophagosome both in yeast and mammals. The class III phosphatidylinositol 3-kinase, PIK3C3C in humans or Vps34 in yeast, produces PtdIns3P by phosphorylating the 3'-OH position of phosphatidylinositol (PtdIns). In order to synthesize PtdIns3P for the initiation of autophagy, PIK3C3/Vps34 has a heterotetrameric core, the PIK3C3 complex I (hereafter complex I) composed of PIK3C3/Vps34, PIK3R4/Vps15, BECN1/Vps30, and ATG14/Atg14. A fifth component of complex I, NRBF2 in mammals and Atg38 in yeast, was found and has been characterized in the past decade. The field has been expanding from cell and structural biology to mouse model and cohort studies. Here I will summarize the structures and models of complex I binding NRBF2/Atg38, its intracellular roles, and its involvement in health and disease. Along with this expansion of the field, different conclusions have been drawn in several topics. I will clarify what has and has not been agreed, and what is to be clarified in the future.

Keywords: atg38; membranes; nrbf2; pik3c3; ptdins3p; vps34.

Figure 1.

Schematic structural models of mutually exclusive complex I (left) and complex II (right). PIK3C3/Vps34, PIK3R4/Vps15, and BECN1/Vps30 are found in both complexes, whereas ATG14/Atg14 is complex I-specific, and UVRAG/Vps38 is complex II-specific. Complex I associates with membranes via the aromatic finger 1 in the BECN1 BARA domain and the BATS domain in ATG14 (which does not exist in Atg14). Complex II associates with membranes via two aromatic finger motifs (aromatic fingers 1 and 2) and the hydrophobic loop, all of which are in the BECN1 BARA domain. C2: C2 domain; CC: Coiled-coil; BARA: BARA domain; BATS: BATS domain; NTD: N terminal domain; CTD: C terminal domain; CXXCs: CXXC motifs; WD40: WD40 domain

Figure 2.

Structures of NRBF2/Atg38. left: schematic representations of NRBF2 and its isoforms (in human residue numbers). The S113 and S120 residues in the intrinsically disordered region (IDR) are phosphorylated by MTOR. Right: structures of the NRBF2 MIT domain (PDB: 4ZEY) and Atg38 coiled-coil domain (PDB: 5KC1). NRBF2/Atg38 makes a stable homodimer via the two segments, the cap and stalk in the coiled-coil domain

Figure 3.

Binding stoichiometry between complex I and NRBF2/Atg38. In mammals, the stoichiometry between complex I and NRBF2 is 1:2 (one copy of complex I and one homodimer of NRBF2, left) when NRBF2 is more abundant than complex I, whereas it can be 2:2 (two copies of complex I and one homodimer of NRBF2, right) when complex I is more abundant than NRBF2. In yeast the stoichiometry is always 1:2 regardless of the comparative abundance between complex I and Atg38. It must be noted that the complex I subunit (S) binding to the MIT domain is still speculative

Figure 4.

Autophagy and endocytic pathways that involve NRBF2. Autophagy: In nutrient-rich conditions, MTORC1 inhibits autophagy by phosphorylating the ULK1 complex and complex I-NRBF2 complex. Upon starvation, MTORC1 is inactivated, leading to the ULK1 complex activation that recruits complex I-NRBF2, which in turn produces PtdIns3P (1. Inititation). PtdIns3P recruits its effectors such as DFCPs and WIPIs at omegasomes that causes nucleation of phagophores (2. Nucleation). The phagophore expands (3. Expansion), then closes (4. Closure) to become an autophagosome. The autophagosome fuses with lysosomes to become an autolysosome (5. Maturation). The cargos are eventually degraded in the autolysosome (6. Degradation). As well as the initiation step, NRBF2 is also involved in the autophagy maturation step by associating with the MON1-CCZ1 complex. The NRBF2-MON1-CCZ1 facilitates the GEF activity of RAB7, which is involved in the maturation step. Circled P: phosphorylation; ?: the direct involvement of NRBF2 has not been confirmed. Endocytic pathway: During endocytosis, lipids and surface proteins including ligand-receptor complexes are internalized. The endocytosed vesicle first fuses with the early endosome marked by RAB5. The early endosome matures into the late endosome marked by RAB7, then the late endosome eventually fuses with lysosomes to degrade the endocytosed cargos. The early to late endosome transition is mediated by the MON1-CCZ1 complex. The CORVET and HOPS complexes facilitate the tethering of early endosomes and late endosomes, respectively. NRBF2 was found to interact with VPP33A, the common subunit between the CORVET and HOPS complexes. Also, NRBF2 is known to interact with the MON1-CCZ1 complex during autophagy and phagocytosis, but it remains to be seen whether this interaction also occurs in the endocytic pathway or not. The complex II-specific UVRAG subunit is also known to bind to the HOPS complex independently of complex II. It has been unclear whether the NRBF2 and UVRAG interactions with the HOPS complex are mutually exclusive or not

Figure 5.

Phagocytosis and LAP pathways where PIK3C3 complexes and NRBF2 are involved. In phagocytosis, the closed phagosome first fuses with early endosomes to become the early phagosome marked by RAB5. The early phagosome matures into the late phagosome, and it eventually fuses with lysosomes to become the phagolysosome (maturation), then the particle is degraded. Similar to the endocytic pathway, RAB5 recruits complex II to the early phagosome, and the RAB5-RAB7 transition is mediated by the MON1-CCZ1 complex. In LAP, complex II and RUBCN/Rubicon are recruited to the closed phagosome (LAPosome, step1), where RUBCN is essential for the recruitment of complex II. In contrast to phagocytosis, the complex II- RUBCN complex is required for the recruitment of RAB5 [74]. The PtdIns3P synthesized by the complex II- RUBCN complex recruits the NOX2 complex, which synthesizes ROS from NADPH on the LAPosome (step2). This is followed by LC3 lipidation (step 3). Along with LC3-II [74], RAB7 activated by the MON1-CCZ1 complex facilitates LAPosome fusion with lysosomes (maturation) to degrade the particle. In BMDM from mice, NRBF2 is required for the maturation step of LAP by facilitating the GEF activity of the MON1-CCZ1 complex to activate RAB7