Abstract
Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.
Highlights
Clathrin-mediated endocytosis is a multi-step process of cargo internalization from the plasma membrane that is essential for the regulation of cell receptor density and uptake of nutrients essential for cell function [1]
We have examined the requirement for PtdIns(4,5)P2 in apical and basolateral endocytosis in polarized kidney epithelial cells by testing the effects of overexpressing phosphatidylinositol 4-phosphate 5kinases type I (PIP5KI) isoforms on these pathways
To examine the distribution and role of PIP5KI isoforms in murine cortical collecting duct (mCCD) cells, we generated DOX-repressible replication-deficient adenoviruses expressing each PIP5KI isoform tagged C-terminally with the HA epitope. mCCD cells were co-infected with varying multiplicity of infection (MOI) of adenovirus together with adenovirus constitutively expressing the tetracycline transactivator, which is required for expression of PIP5KIs driven by the tet operon
Summary
Clathrin-mediated endocytosis is a multi-step process of cargo internalization from the plasma membrane that is essential for the regulation of cell receptor density and uptake of nutrients essential for cell function [1]. The transferrin receptor mediates internalization of iron loaded transferrin from the cell surface to maintain iron homeostasis. The incorporation of these and other diverse cargoes into forming clathrin-coated pits (CCPs) is facilitated by endocytic adaptors proteins, including AP2, epsin, autosomal recessive hypercholesterolemia (ARH), and disabled 2 (Dab2) [3]. The lipid phosphatidylinositol 4,5bisphosphate [PtdIns(4,5)P2] plays a key role in recruitment of clathrin adaptors and other regulatory proteins critical for endocytosis to the plasma membrane [5]
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