Beclin-1-Associated Autophagy Related Key Regulator (Barkor)

Mutation analysis revealed that both coiled-coil domains of Atg14 were required for the interaction of Atg14 with beclin-1 and Vps34. Gel filtration experiments revealed that endogenous Vps34, beclin-1, Atg14, and Rubicon coeluted in a complex of over 700 kD. However, immunoprecipitation analysis showed that Atg14 and Rubicon did not always coimmunoprecipitate in the same complex. Knockdown of Atg14 or beclin-1 via small interfering RNA impaired autophagy-mediated clearance of a test substrate. Knockdown of Atg14 in mouse fibroblasts resulted in accumulation of large puncta representing ubiquitinated protein inclusions and reduced the number of autophagosomes. Atg14 also stimulated Vps34 kinase activity, but only when coexpressed with beclin-1.