Techniques for Studying the Secretory Pathway

The key to understanding how proteins are transported through the organelles of the secretory pathway has been to develop a basic description of the function of transport vesicles. Many components required for the formation and fu sion of transport vesicles have been identified in the past decade by a remarkable convergence of the genetic and biochemical approaches described in this section. All studies of intracellular protein trafficking employ some method for assaying the transport of a given protein from one compartment to another. We begin by describing how intracellular protein transport can be followed in living cells and then consider genetic and in vitro systems that have proved useful in elucidating the secretory pathway.

Transport of a Protein Through the Secretory Pathway Can Be Assayed in Living Cells

The classic studies of G. Palade and his colleagues in the 1960s first established the order in which proteins move from organelle to organelle in the secretory pathway. These early studies also showed that secretory proteins were never released into the cytosol, the first indication that transported proteins are associated with some type of membrane-bounded intermediate. In these experiments, which combined pulse-chase labeling (see Figure 3-36) and autoradiography, radioactively labeled amino acids were injected into the pancreas of a hamster. At different times after injection, the animal was sacrificed and the pancreatic cells were chemically fixed, sectioned, and subjected to autoradiography to visualize the location of the radiolabeled proteins. Because the radioactive amino acids were administered in a short pulse, only those proteins synthesized immediately after injection were labeled, forming a distinct group, or cohort, of labeled proteins whose transport could be followed. In addition, because pancreatic acinar cells are dedicated secretory cells, almost all of the labeled amino acids in these cells are incorporated into secretory proteins, facilitating the observation of transported proteins.

Although autoradiography is rarely used today to localize proteins within cells, these early experiments illustrate the two basic requirements for any assay of intercompartmental transport. First, it is necessary to label a cohort of proteins in an early compartment so that their subsequent transfer to later compartments can be followed with time. Second, it is necessary to have a way to identify the compartment in which a labeled protein resides. Here we describe two modern experimental procedures for observing the intracellular trafficking of a secretory protein in almost any type of cell.

In both procedures, a gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomatitis virus (VSV) is introduced into cultured mammalian cells either by transfection or simply by infecting the cells with the virus. The treated cells, even those that are not specialized for secretion, rapidly synthesize the VSV G protein on the ER like normal cellular secretory proteins. Use of a mutant encoding a temperature-sensitive VSV G protein allows researchers to turn subsequent protein transport on and off. At the restrictive temperature of 40 °C, newly made VSV G protein is misfolded and therefore retained within the ER by quality control mechanisms discussed in Chapter 16, whereas at the permissive temperature of 32 °C, the accumulated protein is correctly folded and is transported through the secretory pathway to the cell surface. This clever use of a temperature-sensitive mutation in effect defines a protein cohort whose subsequent transport can be followed.

In two variations of this basic procedure, transport of VSV G protein is monitored by different techniques. Studies using both of these modern trafficking assays and Palade's early experiments all came to the same conclusion: in mammalian cells vesicle-mediated transport of a protein molecule from its site of synthesis on the rough ER to its arrival at the plasma membrane takes from 30 to 60 minutes.

Microscopy of GFP-Labeled VSV G Protein One approach for observing transport of VSV G protein employs a hybrid gene in which the viral gene is fused to the gene encoding green fluorescent protein (GFP), a naturally fluorescent protein (Chapter 5). The hybrid gene is transfected into cultured cells by techniques described in Chapter 9. When cells expressing the temperature-sensitive form of the hybrid protein (VSVG-GFP) are grown at the restrictive temperature, VSVG-GFP accumulates in the ER, which appears as a lacy network of membranes when cells are observed in a fluorescent microscope. When the cells are subsequently shifted to a permissive temperature, the VSVG-GFP can be seen to move first to the membranes of the Golgi apparatus, which are densely concentrated at the edge of the nucleus, and then to the cell surface (Figure 17-2a). By analyzing the distribution of VSVG-GFP at different times after shifting cells to the permissive temperature, researchers have determined how long VSVG-GFP resides in each organelle of the secretory pathway (Figure 17-2b).

Detection of Compartment-Specific Oligosaccharide Modifications A second way to follow the transport of secretory proteins takes advantage of modifications to their carbohydrate side chains that occur at different stages of the secretory pathway. To understand this approach, recall that many secretory proteins leaving the ER contain one or more copies of the A-linked oligosaccharide Man8(GlcNAc)2, which are synthesized and attached to secretory proteins in the ER (see Figure 16-18). As a protein moves through the Golgi complex, different enzymes localized to the cis-, medial-, and trans-Golgi cisternae catalyze an ordered series of reactions to these core Man8(GlcNAc)2 chains. For instance, glycosidases that reside specifically in the cis-Golgi compartment sequentially trim mannose residues off of the core oligosaccharide to yield a "trimmed" form Man5(GlcNAc)2 (Figure 17-3, reaction 1). Scientists can use a specialized carbohydrate-cleaving enzyme known as endoglycosidase D to distinguish glycosylated proteins that remain in the ER from those that have entered the cis-Golgi: trimmed cis-Golgi-specific oligosaccharides are cleaved from proteins by endoglycosidase D, whereas the core (untrimmed) oligosaccharide chains on secretory proteins within the ER are resistant to cleavage by this enzyme. Because a deglycosylated protein produced by endoglycosidase D digestion moves faster on an SDS gel than the corresponding glycosylated protein, they can be readily distinguished.

This type of assay can be used to track movement of VSV G protein in virus-infected cells pulse-labeled with radioactive amino acids. Immediately after labeling, all the extracted labeled VSV G protein is still in the ER and is resistant to digestion by endoglycosidase D, but with time an increasing fraction of the glycoprotein becomes sensitive to digestion

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