Ent3p is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body

Ptdins(3,5)P-2 is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P-2 effector localized to endosomes. The ENTH domain of Ent3p is essential for its Ptdins(3,5)P-2 binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P-2 binding via its ENTH domain.