Formation and function of a novel Atg21-retromer complex in

Atg18, Atg21 and Hsv2 are homologous proteins that fulfill macroautophagic/autophagic and non-autophagic functions. We now found that Atg21 interacts with Pep8/Vps26, Vps29 and Vps35, the components of the cargo selective complex of the retromer. We identified Atg21 residues required for retromer binding and focused on two of them. The first, T106, is part of an STS-motif, which also mediates Atg18-binding to the retromer, while in Hsv2 this motif is not conserved. As a second retromer binding residue, we identified D28 of Atg21. Interestingly, the corresponding D45 of Hsv2 also confers retromer binding, but the analogous E34 of Atg18 does not. Together, Atg18 uses binding residue 1, while Atg21 uses 1 and 2 and Hsv2 only 2. During autophagy, Atg21 organizes the Atg8-lipidation machinery by interacting with Atg16 via the bottom side of its β-propeller. Partial overlap between the Atg16 binding residues and the retromer binding residues indicates mutually exclusive interaction. Indeed, lack of Atg16 enhances Atg21 binding to the retromer. The Atg21-retromer shows vacuole fission activity, which requires both retromer binding residues and the membrane-bending activity of its loop 6 C/D. Additionally, overexpression of Atg21 led to mislocalization of the Prc1/carboxypeptidase Y cargo receptor Pep1/Vps10 from the Golgi to Vps17-positive endosomes and to Prc1 secretion. We detected a cross-talk among the different retromer complexes. In the absence of the canonical retromer component Vps5, more Atg21-retromer complexes were formed. Furthermore, the vacuole hyper-fragmentation of