Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicul...

Reconstitution of nuclear protein transport with semi-intact yeast cells

We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mam...

Geranylgeranylation but Not GTP Loading

Differential Regulation of Exocytosis by Calcium and CAPS in Semi-Intact Synaptosomes

Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca2+-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from...

Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells.

We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, alth...