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

Abstract

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 transGolgi 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 organdies 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 ATE and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP3,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 Ca 2÷ 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.

Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane

“…Synaptophysin is sulfated on carbohydrate residues Sulfation of proteins on tyrosine and carbohydrate residues, both of which occur selectively in the TGN (Kimura et al, 1984;Baeuerle and Huttner, 1987), has been found to be a powerful tool to study protein exit from the TGN (Tooze and Huttner, 1990;Miller and Moore, 1991 (Wiedenmann and Franke, 1985) from PC12 cells after a 5 min pulse with methionine followed by a 2 h chase ( Figure 1, (Figure 2A,B,G; see also Tooze and Huttner, 1900 . , .…”

Sorting and processing of secretory proteins

“…Release of preformed products from the regulated pathway is further distinguished by being dependent upon protein synthesis, whereas the constitutive pathway is not [43]. The generation of both constitutive vesicles and secretory granules of the regulated pathway is, however, GTP-dependent [44][45][46].…”

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

“…The method of Miller and Moore (1991) for permeabilizing cells with SLO was Vol. 6, February 1995 modified slightly and used to allow BFA direct access to the cytosol of wild-type or mutant CHO cells.…”