The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression.

The class III phosphatidylinositol 3-kinase (PI3KC3) plays a central role in autophagy. Rubicon, a RUN domain-containing protein, is newly identified as a PI3KC3 subunit through its association with Beclin 1. Rubicon serves as a negative regulator of PI3KC3 and autophagosome maturation. The molecular mechanism underlying the PI3KC3 and autophagy inhibition by Rubicon is largely unknown. Here, we demonstrate that Rubicon interacts with the PI3KC3 catalytic subunit hVps34 via its RUN domain. The RUN domain contributes to the efficient inhibition of PI3KC3 lipid kinase activity by Rubicon. Furthermore, a Rubicon RUN domain deletion mutant fails to complement the autophagy deficiency in Rubicon-depleted cells. Hence, these results reveal a critical role of the Rubicon RUN domain in PI3KC3 and autophagy regulation.