Showing papers by "Markus R. Wenk published in 2003"

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ

Abstract: Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPγS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Iγ (PIPKIγ), in this effect. These data suggest a model according to which activation of PIPKIγ by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry

Abstract: Phosphoinositides (phosphorylated derivatives of phosphatidylinositol, PI) are versatile intracellular signaling lipids whose occurrence in low concentrations complicates direct mass measurements. Here we present a sensitive method to detect, identify and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)) with different fatty acid compositions (phosphoinositide profiles) in total lipid extracts by electrospray ionization mass spectrometry (ESI-MS). Using this method, we detected elevated concentrations of PIP2 in human fibroblasts from patients with Lowe syndrome, a genetic disorder that affects phosphoinositide metabolism. Saccharomyces cerevisiae cells deficient in enzymes involved in PIP metabolism--Sac1p, a phosphoinositide phosphatase, and Vps34p and Pik1p, a PI 3-kinase and PI 4-kinase, respectively--showed not only different PIP concentrations but also differential changes in PIP profiles indicating metabolic and/or subcellular pooling. Mass spectrometric analysis of phosphoinositides offers unique advantages over existing approaches and may represent a powerful diagnostic tool for human diseases that involve defective phosphoinositide metabolism.

Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

Abstract: Phosphorylation of inositol phospholipids plays a key role in cellular regulation via the generation of intracellular second messengers. In addition, it represents a mechanism to regulate interactions of the lipid bilayer with proteins and protein scaffolds involved in vesicle budding, cytoskeletal organization, and signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from phosphatidylinositol (PI) is an important step in this metabolic pathway because PI(4)P is a precursor of other important phosphoinositides and has protein binding properties of its own. We report here that a PI 4-kinase (PI4K) activity previously reported on synaptic vesicles is accounted for by the α isoform of the recently characterized type II PI4K (PI4KII) family. PI4KIIα, which also accounts for the bulk of PI4K activity in brain extracts, is concentrated at synapses and in the region of the Golgi complex in neuronal perikarya. Our results provide new evidence for the occurrence of a cycle of phosphoinositide synthesis and hydrolysis nested within the exo–endocytic cycle of synaptic vesicles and point to PI4KIIα as a critical player in this cycle.

The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein- dependent pathway

Abstract: Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TG...

Assembly of endocytosis-associated proteins on liposomes.

Abstract: Publisher Summary This chapter describes the methodology of a cell-free system utilizing liposomes and cytosol to study the assembly reactions of proteins involved in clathrin-mediated endocytosis. Liposomes, when incubated with brain cytosol and nucleotides, undergo morphological changes and fragmentation into smaller structures that resemble closely intermediates of clathrin-mediated endocytosis. The biochemical and biophysical analysis of these reactions show that the elevated levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 , or PIP 2 ] correlate with increased recruitment of clathrin and clathrin adaptors to the liposomal surface. Inclusion into the liposomes of a surface-exposed recombinant fragment of synaptotagmin enhances the recruitment of clathrin coat proteins, thereby demonstrating the synergistic actions of proteins and lipids. Three modes of analysis are applied in the study described in the chapter: (1) observation of liposomal structure by electron microscopy, (2) protein biochemistry to assess the amounts of endocytotic proteins recruited to the liposomal surface, and (3) lipid biochemistry to measure the metabolism of phosphoinositides during the incubation reaction. Functionalized liposomes, such as the proteoliposomes, which areideally suited to investigate the synergistic effect of membrane lipids and membrane proteins on coat structure and function, are also described.