Phosphatidylinositol 4-Kinase IIβ Associates with Clathrin Coated Vesicles in Living Cells as Revealed by Brightness Analysis

Abstract

Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated Types II and III, which phosphorylate phosphatidylinositol to generate PI4P. A number of studies indicate that these enzymes are important for Golgi trafficking and early as well as late stages of endocytosis. In this study, we focus on PI4KIIβ, a protein that is evenly distributed between membrane and soluble fractions and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIβ exists as two distinct species in the cytoplasm, a soluble monomer and a high order complex enriched with multiple copies of PI4KIIβ. This observation is confirmed by autocorrelation analysis which identifies two species with distinct mobilities. We further demonstrate that the high order complex enriched with PI4KIIβ is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high order PI4KIIβ complex is sensitive to expression of dynamin 2-K44A, a dominant-negative inhibitor of endocytosis. We further directly detect that PI4KIIβ co-moves with clathrin light chain on vesicles using dual-color heterospecies partition analysis. This analysis allows us to isolate the co-mobile species in the presence of strong background contribution from the monomeric pool of PI4KIIβ. Our results strongly suggest that PI4KIIβ is involved in an early stage of endocytosis, and associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool to characterize cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques. This work is supported by the National Institutes of Health (R01 GM64589) and the National Science Foundation (PHY-0346782).