title: Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.
abstract: 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 (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720.
authors.forename: S G
authors.initials: SG
authors.lastname: Miller
authors.forename: H P
authors.initials: HP
authors.lastname: Moore
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology*; D002463:Cell Membrane Permeability; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006056:Golgi Apparatus / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000502:physiology*; D066298:In Vitro Techniques; D008561:Membrane Fusion / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000378:metabolism; D015388:Organelles / Q000648:ultrastructure; D013301:Streptolysins; D014759:Viral Envelope Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C028391:G protein, vesicular stomatitis virus; D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D013301:Streptolysins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C025964:streptolysin O; C013786:A73025; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002118:Calcium