Trem2 recycling was impaired in beclin 1-deficient BV2 cells as was observed with CD36 ( Figure 3H). Taken together, these data suggest that beclin 1 has a function in phagocytic receptor recycling. While receptor recycling is regulated by numerous mechanisms, a recent study in C. elegans showed that clearance of apoptotic cell corpses by the phagocytic receptor CED-1 was dependent on receptor recycling via DAPT the retromer complex ( Chen et al., 2010). This conserved structure is responsible

for endosome-to-Golgi retrograde transport of membrane proteins and in mammals consists of the subunits Vps26, Vps29, Vps35, and sorting nexins ( Bonifacino and Hurley, 2008). To determine whether beclin 1 might regulate the retromer complex, we knocked down beclin 1 expression using a lentivirus encoding beclin 1 shRNA and subsequently investigated whether the retromer complex was affected by beclin 1 knockdown. Remarkably, beclin 1 knockdown resulted in a prominent reduction of the retromer complex ( Figures 4A and 4B). To test whether diminished retromer expression impairs CD36 recycling, we knocked down Vps35 levels in BV2 cells using lentivirus encoding

Vps35 shRNA ( Figure 4C) and analyzed CD36 recycling. Reducing Vps35 significantly impaired CD36 recycling ( Figure 4D). Furthermore, reducing Vps35 also resulted in a concomitant reduction in phagocytic efficiency ( Figure 4E). Because previous studies have demonstrated that retromer dysfunction can be reversed by enhancing Vps35 levels ( MacLeod et al., 2013), we rescued Vps35 levels in beclin 1-deficient CB-839 in vitro BV2 cells. Rescuing Vps35 resulted in enhanced CD36 recycling ADAMTS5 ( Figure 4G) and phagocytosis ( Figure 4H). Together these data suggest that beclin 1 regulates the retromer complex, which is required to maintain

phagocytic receptor recycling and phagocytosis. To further explore this connection, we first tested whether beclin 1 might regulate retromer via a direct interaction. This proved unlikely, as we were unable to coimmunoprecipitate beclin 1 and Vps35 (data not shown). Moreover, a recent study using mass spectrometry screens to identify putative binding partners of mammalian beclin 1 and other known autophagy proteins that complex with beclin 1 did not reveal any binding partners exclusive to the retromer complex (Behrends et al., 2010). Therefore, we next tested whether beclin 1 may have a role in the recruitment of retromer. Retromer is typically recruited to vesicles via its sorting nexin subunits. These subunits contain a Phox homology domain capable of binding to phosphatidylinositol 3-phosphate (PI3P) present on target membranes (Burda et al., 2002). Interestingly, phosphatidylinositol is converted to PI3P primarily by the PI3K, Vps34, which is known to form a complex with beclin 1 and regulate autophagy and apoptosis (Funderburk et al., 2010).