Abstract
A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.
Highlights
Characterization of the reconstituted system has revealed that the activity of at least one cytosolic factor is required for endocytic vesicle fusion: this function is lost upon treatment of cytosol with the alkylating agent NEM (3-6)
An early stage of the endocytic pathway involves the hydrolysis of GTP, perhaps due to the involvement of a r&-like protein in vesicle targeting
Other activities one could envision for such factors would be in the uncoating of vesicles prior to the fusion reaction itself
Summary
We have observed that a monoclonal antibody raised against NSF only partially inhibits in vitro fusion of either CHO or K562 endocytic vesicles: at a final concentration of 0.1 mg of antibody/mg of protein in the cell-free assay (a large excess) only 25-30% inhibition in NEM-sensitive In Vitro Endocytic Vesicle Fusion Is Inhibited by Nonhydrolyzable Nucleotide Analogs-A role for GTP hydrolysis during intracellular membrane vesicle transfer has recently been characterized.
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