NRBF2 regulates macroautophagy as a component of Vps34 Complex I

Abstract

Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG- 14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP–LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.

Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

“…The human ortholog of Atg38 is NRBF2 (nuclear receptor binding factor 2). In parallel with the observations for Atg38 and yeast PI3KC3-C1, NRBF2 is an interactor and/or component of the mammalian PI3KC3-C1 complex (15)(16)(17). Cells from NRBF2 knockout mice show reduced autophagy, reduced PI3KC3 activity, and reduced stability of PI3KC3-C1 (15).…”

“…Cells from NRBF2 knockout mice show reduced autophagy, reduced PI3KC3 activity, and reduced stability of PI3KC3-C1 (15). Knockdown of NRBF2 with shRNA resulted in similar effects (16). A third report also concluded that NRBF2 was associated with PI3KC3-C1; however, siRNA knockdown of NRBF2 led to an increase in autophagy in this report (17).…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…160 Intriguingly, in yeast Atg38 is responsible for the stability of Vps34 complex I, which connects Vps34-Vps15 with Atg14-Vps30, 160 reminiscent of the function of NRBF2 in mediating ATG14, PIK3C3-PIK3R4, and BECN1 interaction in mammalian cells. 82 However, it is a question under debate whether NRBF2 positively 81,83 or negatively 82 regulates autophagy in higher eukaryotes.…”

“…61 NRBF2 was identified as a protein involved in autophagy, which is present only in the ATG14-containing class III PtdIns3K complex in mammalian cells. [81][82][83]159 The yeast ortholog of NRBF2 is presumed to be Atg38, although there are sequence diversities. 82,160 Likewise, Atg38 is recognized as a component of the Atg14-containing Vps34 complex I in yeast, which is required for the autophagy pathway.…”

“…2). The stratification can be viewed based on their interaction and stability: PIK3C3-PIK3R4-BECN1, the most stable and prevalent complex, constitutes the core; while ATG14 and UVRAG (UV radiation resistance associated) are combined with the core in PtdIns3K complex I (PtdIns3K-C1; ATG14-containing PtdIns3K complex) and PtdIns3K complex II (PtdIns3K-C2; UVRAG-containing PtdIns3K complex), respectively; 75-78 KIAA0226/Rubicon is a regulatory subunit that is thought to stably bind to UVRAGcontaining complexes; 77,78 AMBRA1, NRBF2 (nuclear receptor binding factor 2) and other accessory factors transiently or unstably interact with either BECN1 (e.g., AMBRA1) 79,80 or ATG14 (e.g., NRBF2), [81][82][83] thus comprising the peripheral components. 84,85 UVRAG also exists in a separate complex excluding ATG14, which holds an important role in the regulation of autophagy at the maturation step.…”