PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization

Abstract

The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P2 dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P2 levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Δ sjl2Δ mutant, which has elevated PtdIns(4,5)P2 levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P2 turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P2 turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.

A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique

“…3C). This was consistent with previous results (26,27). The labeling in the coated pit was also intense at the rim or in the zone of 70-100 nm from the center (Fig.…”

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

“…Because the N-terminal region of Sla2 that contains the ANTH domain [Sla2(1-360)] can support endocytosis in an otherwise wild-type strain (25) and localizes to endocytic sites independently of epsins (Fig. S1D), we tested if Sla2(1-360) is responsible for anchoring Ent1-ENTH to the endocytic site.…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…It seems unlikely that localized PIP2 production contributes to CCP initiation or maturation because no PIP2-kinases have been localized to CCPs (Sun et al 2007;Antonescu et al 2010). In contrast a variety of PIP 2 (5 0 )-phosphatases (OCRL, Sjn1, and Sjn2) do local-ize to CCPs and play important roles in CCP maturation and budding (Perera et al 2006;Mao et al 2009;Nakatsu et al 2010Nakatsu et al , 2012Taylor et al 2011).…”