Yeast Eps15-like endocytic protein Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton

The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles. In yeast, endocytic vesicles move towards early endosomes along actin cables, however, the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood. The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases. Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines, pan1-18TA, displayed almost the same endocytic defect as ark1Δ prk1Δ cells, and contained abnormal actin concentrations including several endocytic compartments. Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables. The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables, and between endocytic vesicles and endosomes. Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables.