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▲ FIGURE 17-3 Processing of W-linked oligosaccharide chains on glycoproteins within cis-, medial-, and trans-Golgi cisternae in vertebrate cells. The enzymes catalyzing each step are localized to the indicated compartments. After removal of three mannose residues in the c/s-Golgi (step 1), the protein moves by cisternal progression to the medial-Golgi. Here, three GlcNAc residues are added (steps 2| and 4|), two more mannose residues are removed (step 3), and a single fucose is added (step 5). Processing is completed in the trans-Golgi by addition of three galactose residues (step 6) and finally by linkage of an ZV-acetylneuraminic acid residue to each of the galactose residues (step 7|). Specific transferase enzymes add sugars to the oligosaccharide, one at a time, from sugar nucleotide precursors imported from the cytosol. This pathway represents the Golgi processing events for a typical mammalian glycoprotein. Variations in the structure of N-linked oligosaccharides can result from differences in processing steps in the Golgi. [See R. Kornfeld and S. Kornfeld, 1985, Ann. Rev. Biochem. 45:631.]

(Figure 17-4). This conversion of VSV G protein from an en-doglycosidase D-resistant form to an endoglycosidase D-sensitive form corresponds to vesicular transport of the protein from the ER to the cis-Golgi. Note that transport of VSV G protein from the ER to the Golgi takes about 30 minutes as measured by either the assay based on oligosaccharide processing or fluorescence microscopy of VSVG-GFP.

▲ EXPERIMENTAL FIGURE 17-4 Transport of a membrane glycoprotein from the ER to the Golgi can be assayed based on sensititivity to cleavage by endoglycosidase D. Cells expressing a temperature-sensitive VSV G protein (VSVG) were labeled with a pulse of radioactive amino acids at the nonpermissive temperature so that labeled protein was retained in the ER. At periodic times after a return to the permissive temperature of 32 °C, VSVG was extracted from cells and digested with endoglycosidase D, which cleaves the oligosaccharide chains from proteins processed in the cis-Golgi but not from proteins in the ER. (a) SDS gel electrophoresis of the digestion mixtures resolves the resistant, uncleaved (slower migrating) and sensitive, cleaved (faster migrating) forms of labeled VSVG. As this electrophoretogram shows, initially all of the VSVG was resistant to digestion, but with time an increasing fraction is sensitive to digestion, reflecting protein transported from the ER to the Golgi and processed there. In control cells kept at 40 °C, only slow-moving, digestion-resistant VSVG was detected after 60 minutes (not shown). (b) Plot of the proportion of VSVG that is sensitive to digestion, derived from electrophoretic data, reveals the time course of ER ^ Golgi transport. [From C. J. Beckers et al., 1987, Cell 50:523.]

Yeast Mutants Define Major Stages and Many Components in Vesicular Transport

The general organization of the secretory pathway and many of the molecular components required for vesicle trafficking are similar in all eukaryotic cells. Because of this conservation, genetic studies with yeast have been useful in confirming the sequence of steps in the secretory pathway and in identifying many of the proteins that participate in vesicular traffic. Although yeasts secrete few proteins into the growth medium, they continuously secrete a number of enzymes that remain

▲ EXPERIMENTAL FIGURE 17-5 Phenotypes of yeast sec mutants identified stages in the secretory pathway. These temperature-sensitive mutants can be grouped into five classes based on the site where newly made secreted proteins (red dots) accumulate when cells are shifted from the permissive temperature to the higher nonpermissive one. Analysis of double mutants permitted the sequential order of the steps to be determined. [See P Novick et al., 1981, Cell 25:461, and C. A. Kaiser and R. Schekman, 1990, Cell 61:723.]

localized in the narrow space between the plasma membrane and the cell wall. The best-studied of these, invertase, hydro-lyzes the disaccharide sucrose to glucose and fructose.

A large number of yeast mutants initially were identified based on their ability to secrete proteins at one temperature and inability to do so at a higher, nonpermissive temperature. When these temperature-sensitive secretion (sec) mutants are transferred from the lower to the higher temperature, they accumulate secreted proteins at the point in the pathway blocked by the mutation. Analysis of such mutants identified five classes (A-E) characterized by protein accumulation in the cytosol, rough ER, small vesicles taking proteins from the ER to the Golgi complex, Golgi cisternae, or constitutive secretory vesicles (Figure 17-5). Subsequent characterization of sec mutants in the various classes has helped elucidate the fundamental components and molecular mechanisms of vesicle trafficking that we discuss in later sections.

To determine the order of the steps in the pathway, researchers analyzed double sec mutants. For instance, when yeast cells contain mutations in both class B and class D functions, proteins accumulate in the rough ER, not in the Golgi cisternae. Since proteins accumulate at the earliest blocked step, this finding shows that class B mutations must act at an earlier point in the secretory pathway than class D mutations do. These studies confirmed that as a secreted protein is synthesized and processed it moves sequentially from the cytosol ^ rough ER ^ ER-to-Golgi transport vesicles ^ Golgi cisternae ^ secretory vesicles and finally is exocytosed.

Cell-free Transport Assays Allow Dissection of Individual Steps in Vesicular Transport

In vitro assays for intercompartmental transport are powerful complementary approaches to studies with yeast sec mu-

tants for identifying and analyzing the cellular components responsible for vesicular trafficking. In one application of this approach, cultured mutant cells lacking one of the enzymes that modify A-linked oligosaccharide chains in the Golgi are infected with vesicular stomatitis virus (VSV). For example, if infected cells lack A-acetylglucosamine trans-ferase I, they produce abundant amounts of VSV G protein but cannot add A-acetylglucosamine residues to the oligosaccharide chains in the medial-Golgi as wild-type cells do (Figure 17-6a). When Golgi membranes isolated from such mutant cells are mixed with Golgi membranes from wild-type, uninfected cells, the addition of A-acetylglu-cosamine to VSV G protein is restored (Figure 17-6b). This modification is the consequence of the retrograde vesicular transport of A-acetylglucosamine transferase I from the wild-type medial-Golgi to the cis-Golgi compartment from virally infected mutant cells. Successful intercompartmental transport in this cell-free system depends on requirements that are typical of a normal physiological process including a cytosolic extract, a source of chemical energy in the form of ATP and GTP, and incubation at physiological temperatures.

In addition, under appropriate conditions a uniform population of the retrograde transport vesicles that move A-acetylglucosamine transferase I from the medial- to cis-Golgi can be purified away from the donor wild-type Golgi membranes by centrifugation. By examining the proteins that are enriched in these vesicles, scientists have been able to identify many of the integral membrane proteins and peripheral vesicle coat proteins that are the structural components of this type of vesicle. Moreover, fractionation of the cytosolic extract required for transport in cell-free reaction mixtures has permitted isolation of the various proteins required for formation of transport vesicles and of proteins required for the targeting and fusion of vesicles with appropriate acceptor

Cis-Goigi

Cis-Goigi

"G protein VSV-infected wild-type cells

"G protein VSV-infected wild-type cells

VSV-infected mutant cells

(no N-acetyigiucosamine transferase I)

VSV-infected mutant cells

(no N-acetyigiucosamine transferase I)

Mediai-Goigi

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