Vacuolar protein sorting 30 (Vps30)4
/autophagy-related protein 6 (Atg6) was originally identified by yeast genetic screening as an essential factor for targeting the vacuolar hydrolase, carboxypeptidase Y (CPY), to the vacuole (1
). At that time, yeast genetic screening also identified Vps30 as an essential factor for autophagy (2
), a bulk degradation pathway, in which a double membrane-bound structure, an autophagosome, sequesters a portion of the cytoplasm and delivers it to the vacuole for degradation (4
In yeast, two distinct phosphatidylinositol 3-kinase (PI 3-kinase) complexes (complexes I and II) have been reported (5
), and share the following three proteins between them: Vps34, the sole PI 3-kinase in yeast, Vps15, a putative protein kinase that activates Vps34 (6
), and Vps30. In addition to these three common components, Atg14 and Vps38 are incorporated into complexes I and II, respectively, and contribute to the complex construction by linking the Vps34-Vps15 complex to Vps30 (5
). Autophagy, as well as most yeast genes responsible for autophagy, is conserved in higher eukaryotes including mammals. In mammals, Beclin 1, the orthologue of yeast Vps30, is known to be a key regulator of mammalian autophagy (8
). Beclin 1 forms a complex with mammalian Vps34 (10
), and recent identification of mammalian Atg14 (also called as Atg14L or Barkor) showed that Beclin 1 forms a PI 3-kinase complex whose composition is similar to that of the yeast PI 3-kinase complex I (11
), although Beclin 1 interacts directly with Vps34 (13
). Beclin 1 also forms several distinct PI 3-kinase complexes, some of which contain UVRAG, a putative Vps38 orthologue (11
), and appears to regulate various membrane trafficking events.
In addition to its complex construction, yeast Atg14 was shown to be crucial for targeting of the PI 3-kinase complex I to the pre-autophagosomal structure (PAS), a perivacuolar structure where most of the Atg proteins colocalize and contribute to autophagosome formation (16
). Similarly, mammalian Atg14 was shown to target the mammalian PI 3-kinase complex I to a specific site in the endoplasmic reticulum, termed an omegasome, which is a putative site of autophagosome formation in mammals (19
). Vps38 was shown to be crucial for targeting of the PI 3-kinase complex II to endosomes in yeast (18
). In mammals, UVRAG localizes at endosomes, suggesting that it functions similarly with Vps38 (11
). Phosphatidylinositol 3-phosphates produced at autophagic membranes and endosomes by PI 3-kinase complexes recruit effector proteins to regulate each pathway (21
). Although elucidation of the molecular roles of PI 3-kinase complexes and their components other than Vps30/Beclin 1 is underway, the molecular roles of Vps30/Beclin 1 remain poorly understood.
Thus far, structural study on Vps30/Beclin 1 family proteins has been limited; such research has been restricted to the Bcl-2 homology 3 (BH3) domain (~20-residue fragment) of Beclin 1 as a complex with Bcl-2 family proteins (26
). A coiled-coil domain (CCD) has been predicted to be located at the central region of Vps30/Beclin 1 family proteins, and Beclin 1 CCD was shown to mediate interactions with various target proteins including Atg14 and UVRAG (12
). Residues 244–337 of Beclin 1, which correspond to the C-terminal portion of the CCD and its following ~70-residue region, are highly evolutionarily conserved; therefore, the region was named the evolutionarily conserved domain (ECD) and its significance in autophagy was clarified (30
). Despite the important roles of these conserved domains, their structural information has been totally lacking.
Here, we report the crystal structure of the C-terminal region of Vps30, which overlaps the C-terminal portion of the ECD and the extreme C-terminal region of Vps30. The structure is a novel globular fold comprising three β-sheet-α-helix repeats. In vivo studies have shown that the region is required for autophagy but not for vacuolar protein sorting. Thus, this region is named the β-α repeated, autophagy-specific (BARA) domain. Further analyses demonstrated that BARA is dispensable for the construction of PI 3-kinase complexes, but is crucial for the targeting of complex I to the PAS.