Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

Abstract

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. I n eukaryotic cells, transport vesicles are implicated in a variety of intracellular traffic events in both the secretory and endocytic pathways. The formation of a carrier vesicle is initiated by the recruitment of cytosolic coat proteins to the cytoplasmic surface of the donor membrane. One of the functions of the coat is to provide a scaffold for the formation of a high-curvature membrane bud. The coated bud then is pinched off as a coated vesicle, which eventually sheds the coat before fusion with its acceptor compartment. Several types of coats including the COPI, COPII, and clathrin coats have been characterized extensively (reviewed in refs. 1-7).Recently, the role of lipid factors is becoming a focus of the investigation in the field of membrane-coat interactions (7-11). Growing evidence suggests a key function of phosphoinositides and in particular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] in the recruitment of endocytic coats. PtdIns(4,5)P 2 binds to a variety of proteins that function in endocytic traffic including the ␣-subunit of clathrin adaptor protein (AP)2 (12), AP180, CALM (13), dynamin (14), and epsin (15). Phosphoinositides affect AP2's self-assembly (12, 16) and stimulate dynamin's GTPase activity (17,18). Furthermore, disruption of these interactions in living cells resulted in inhibition of endocytosis (15,19). Strong evidence for a physiological role of phosphoinositide metabolism in endocytosis came from the identification and functional characterization of a synaptic polyphosphoinositide phosphatase, synaptojanin 1 (20). Genetic deletion of synaptojanin in mice (21) and Caenorhabditis elegans (22) or disruption of the interactions of synaptojanin with its functional partners (23) resulted in a partial impairment of synaptic vesicle recycling and an increase of clathrin-coated vesicles and the coated pits. Conversely, an enzyme that synthesizes PtdIns(4,5)P 2 from PtdIns 4-monophosphate [PtdIns (4)P], PtdIns (4)P 5-kinase I␥, was shown recently to be concentrated at the synapse (24). Thus, the PtdIns(4,5)P 2 level at the synapse is likely to result from a balance of the enzymatic activities of synaptojanin and PtdIns (4)P 5-kinase.In vitro reconstitution of specific steps of vesicular transport reactions represents an increasingly important strategy to elucidate molecular mechanisms involved ...

The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature

“…Dynamin‐dependent vesicle formation can be reconstituted in vitro by incubation of large unilamellar liposomes with brain cytosol in the presence of nucleotides, and monitored quantitatively by dynamic light scattering (DLS) (Kinuta et al , 2002). To establish the importance of amphiphysin in vesicle formation, we compared brain cytosol from wild‐type and amphiphysin 1 knockout mice in this assay.…”

“…Brain cytosol from wild‐type mice and amphiphysin 1 knockout mice (Di Paolo et al , 2002) was prepared essentially by the previous method and used (Kinuta et al , 2002). Dynamin 1 was purified from bovine brains by the method of Liu et al (1994).…”

“…Large unilamellar liposomes composed of 80% (w/w) FF and 20% cholesterol in 0.3 M sucrose (1 mg/ml) were prepared as described previously (Takei et al , 1999). Vesicle formation was performed by the method of Kinuta et al (2002), with minor modification. A reaction mixture (500 μl) containing cytosolic buffer (25 mM Hepes–KOH (pH 7.2), 25 mM KCl, 2.5 mM magnecium acetate, 100 mM potassium glutamate), 100 μg of liposomes, brain cytosol from either wild‐type or amphiphysin knockout mice (500 μg/ml protein), 2 mM ATP and 200 μM GTP was incubated at 37°C for 15 min.…”

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

“…For actin polymerization assay, small liposomes of uniform size, ϳ100 nm in diameter, composed of 50% PS/50% PC, 50% phosphatidylethanolamine/50% PC, 50% PI/50% PC, 50% phosphatidic acid/50% PC, or 10% PI(4,5)P 2 /90% PC were used. For this purpose, large unilamellar liposomes in 1 mM EDTA, 20 mM Hepes, pH 7.4, were prepared as described previously (21) and then extruded in a syringe-type extruder (Avanti Polar Lipids) six times through polycarbonate membranes (pore size, 100 nm).…”

PIP5KIγ is required for cardiovascular and neuronal development

“…Phosphoinositide kinase activity in total brain lysate was determined as described previously (40). Lipids were separated by TLC using 70 mM CHCl 3, 100 mM MeOH, 25 mM H 2 O , and 15 mM NH4OH (vol/vol).…”