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W-Acetylglucosamine transferase I reaction

W-Acetylglucosamine transferase I reaction

Trans-Goigi

Golgi isolated from G protein in Golgi from uninfected wild-type cells infected mutant cells

Golgi isolated from G protein in Golgi from uninfected wild-type cells infected mutant cells

Addition of N-acetyi-giucosamine to G protein

■ = N-Acetyigiucosamine

O =

Gaiactose

O = Mannose

o=

N-Acetyineuraminic acid

Addition of N-acetyi-giucosamine to G protein

▲ EXPERIMENTAL FIGURE 17-6 Protein transport from one Golgi cisternae to another can be assayed in a cell-free system. (a) A mutant line of cultured fibroblasts is essential in this type of assay. In this example, the cells lack the enzyme W-acetylglucosamine transferase I (step 2| in Figure 17-3). In wild-type cells, this enzyme is localized to the medial-Golgi and modifies W-linked oligosaccharides by the addition of one W-acetylglucosamine. In VSV-infected wild-type cells, the oligosaccharide on the viral G protein is modified to a typical complex oligosaccharide, as shown in the trans-Golgi panel. In infected mutant cells, however, the G protein reaches the cell surface with a simpler high-mannose oligosaccharide containing only two N-acetylglucosamine and five mannose residues. (b) When Golgi cisternae isolated from infected mutant cells are incubated with Golgi cisternae from normal, uninfected cells, the VSV G protein produced in vitro contains the additional N-acetylglucosamine. This modification is carried out by transferase enzyme that is moved by retrograde transport vesicles from the wild-type medial-Golgi cisternae to the mutant c/s-Golgi cisternae in the reaction mixture. [See W. E. Balch et al., 1984, Cell 39:405 and 525; W. A. Braell et al., 1984, Cell 39:511; and J. E. Rothman and T Söllner, 1997, Science 276:1212.]

membranes. In vitro assays similar in general design to the one shown in Figure 17-6 have been used to study various transport steps in the secretory pathway.

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