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

Abstract

The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell-free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a "donor" to an "acceptor" Golgi fraction. The donor Golgi fraction is prepared from VSV-infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase I. "Acceptor" is prepared from uninfected wild-type CHO cells. Transport is measured by the addition of N-acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non-hydrolyzable GTP analogue, GTP gamma S. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non-clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.

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

“…One protein that has been followed extensively as a processing marker of widespread utility including the liver system is the G protein of VSV (Rothman et al 1984;Balch et al 1987;Wattenberg 1991). To validate endoplasmic reticulum to Golgi apparatus transfer, VSV G protein is radiolabeled and the conversion of the mannose 8-9 form present in transitional endoplasmic reticulum to the mannose 5 form of the Golgi apparatus is monitored.…”

“…Such assays have the advantage of measuring both vesicle attachment and vesicle fusion since the mixing of constituents within the fusing compartments is necessary for signal generation. Examples include the processing of vesicular stomatitis virus G proteins Wattenberg 1991) and the acquisition of specific α1,6-mannose residues by [ 35 S]-labeled core-glycosylated pro-α-factor, the precursor to a secreted mating pheromone in yeast (Salama et al 1993).…”

Segregation of secretory material in all elements of the Golgi apparatus in principal epithellal cells of the rat seminal vesicle

“…The question as t o how such procollagen-containing large cylindrical distensions of the saccules or prosecretory granules migrate from the cis to the trans-aspect of the Golgi stacks is still problematical. This is especially true in view of the current concept that Golgi saccules constitute an immobile phase, whereas glycosylated proteins are vectorially transported in a cistrans direction by means of small vesicles that fuse with and bud off the edges of the saccules (reviews in Dunphy and Rothman, 1985;Farquhar, 1985;Rothman, 1985;Pfeffer and Rothman, 1987;Wattenberg, 1991).…”

Transport of casein submicelles and formation of secretion granules in the golgi apparatus of epithelial cells of the lactating mammary gland of the rat

“…Numerous vesicles of various sizes are seen associated with the stacks of Golgi saccules and these have 0 1993 WILEY-LISS, INC generally been considered as carriers of proteins not only from the proximal cisternae of the endoplasmic reticulum to the cis aspect of the Golgi stacks but also from one saccule to the next in a cis-trans direction until the proteins are packaged within vesicles budding from the trans-most Golgi element or trans-Golgi network (Palade, 1975;Farquhar, 1983Farquhar, , 1985Farquhar and Palade, 1981;Goldfischer, 1982;Rothman, 1981Rothman, , 1985Dunphy and Rothman, 1985;Pfeffer and Rothman, 1987;Griffith and Simons, 1986;Wattenberg, 1991). According to this vesicular transport model, the vesicles would be mobile, while the other elements of the Golgi stacks would be comparatively stable.…”