Intracellular membrane flow: reconstitution of transition vesicle formation and function in a cell-free system.

Abstract

Transfer of membrane between endoplasmic reticulum and Golgi apparatus in situ is considered to occur via 60-nm transition vesicles derived from part-rough, partsmooth transition elements of the endoplasmic reticulum. A procedure is described for the isolation of a fraction enriched in these transition elements from rat liver. The isolated fraction generates small vesicles morphologically resembling transition vesicles when incubated with nucleoside triphosphate at 370C. In the cell-free system consisting of a donor fraction enriched in transition elements and an acceptor fraction consisting of intact Golgi apparatus immobilized on nitrocellulose strips, transfer in vitro of radiolabeled membranes was demonstrated. Nucleoside triphosphates were required for transfer, and transfer was facilitated by a cytosol fraction of Mr >10,000. In the presence of both nucleoside triphosphate and cytosol, radiolabeled proteins were transferred in a manner dependent upon both time and temperature. Transfer appeared to be both vectorial and specific in that, with Golgi apparatus (or endoplasmic reticulum) as both donor and acceptor, only negligible time and temperature-dependent transfer was observed. The test system described is expected to facilitate further investigation of the transfer process and to provide a convenient assay to guide transition vesicle isolation and characterization.Transfer of membrane materials from endoplasmic reticulum to Golgi apparatus long has been considered from morphological evidence to be mediated by transition vesicles that bleb off specialized, part-rough, part-smooth regions of the endoplasmic reticulum (1, 2). These vesicles, covered by a nap-like coat material not containing clathrin (3-5), are thought to coalesce to form new Golgi apparatus cisternae or to fuse with existing Golgi apparatus membranes to effect delivery to the Golgi apparatus of materials derived from the endoplasmic reticulum (6).Operation of the segment of the exocytotic pathway has been assumed largely from static images provided from electron microscopy. To demonstrate the phenomenon in vitro, we used a cell-free incubation mixture patterned after that described by Rothman and colleagues (7,8). With appropriately "primed" (complete incubation, 37°C) preparations of part-rough, part-smooth transitional regions of endoplasmic reticulum from rat liver, we observed the formation of small blebbing profiles similar to those associated with transition elements in situ (9). These vesicles were characterized by an electron-dense (but not clathrin-derived) coat material.In this paper, we report the reconstitution of membrane transfer in a cell-free environment using rat liver fractions. Radioactivity was transferred from transition elements metabolically labeled with [3H]leucine to Golgi apparatus immobilized on nitrocellulose strips. Transfer was time and temperature dependent and was enhanced by the presence of nucleoside triphosphate (plus a regenerating system) and a cytosol fraction of Mr >10,000.MATERIALS AND METHO...

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

“…Completely cellfree systems have also been used to investigate intraGolgi apparatus trafficking (Fries and Rothman 1980;Balch et al 1984a,b;Dunphy and Rothman 1985) and endocytic events (Gruenberg and Howell 1989;Hammond et al 1994). Also studied in completely cell-free systems are transfers between the endoplasmic reticulum and the Golgi apparatus Nowack et al 1987;Paulik et al 1988, Ruohola et al 1988; Wuestehube and Schekman 1992; Barlowe et al 1994;Bannyk et al 1996;Bannyk and Balch 1997).…”

Analysis of protein transport through the golgi in a reconstituted cell‐free system

“…These workers utilize a metabolically labeled subcellular fraction enriched in ER transitional elements (Nowack et al, 1987) or vesicles derived from the transitional elements (Paulick et al, 1988). These fractions are incubated with a Golgi fraction that has been immobilized on nitrocellulose strips.…”

Membrane fusion

“…In permeabilized cells and cellfree reconstitution systems a number of requirements have to be met in order to obtain efficient fusion of intracellular membranes. Some of these requirements are; ATP or GTP, proteinaceous components present in the cytosol and on the cytoplasmic surface of donor and acceptor membranes, and ions 5,7,29,40,59,60,64,98 In general, efficient fusion does not require a rise in Ca 2 + concentration above the physiological 'resting level' (0.1/aM) 7 Again, proteins appear to play a crucial role in the regulation of membrane fusion. A potential role for GTP-binding proteins in the regulation of membrane fusion has been put forward 59'6~ In addition, a protein named NSF (N-ethylmaleimide-sensitive factor) 10, has been identified and shown to be involved in vesicular transport, within the Golgi stack 10, from ER to Golgi 8, and during endocytosis 3o.…”