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 fusion of transport vesicles have been identified by a remark able 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 live 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 Live Cells
The classic studies of G. Palade and his colleagues in the 1960s first established the order in which proteins move from one organelle to the next in the secretory pathway. These early studies also showed that secretory proteins are never released into the cytosol— the first indication that transported proteins are always associated with some type of membrane-bounded intermediate. In these experiments, which combined pulse-chase labeling and autoradiography, radioactively labeled amino acids were injected into the pancreas of hamsters. At different times after injection, the animals were sacrificed and the pancreatic cells were immediately fixed with glutaraldehyde, sectioned, and subjected to autoradiography to visualize the locations 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 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 were 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 intercompart mental 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 over 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 mem brane 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 as they would normal cellular secretory proteins. Use of a mutant gene encoding a temperature-sensitive VSV G protein allows researchers to turn subsequent transport of this protein on and off. At the restrictive temperature of 40 °C, newly made VSV G protein is misfolded and is therefore retained within the ER by the quality-control mechanisms, whereas at the permissive temperature of 32 °C, the protein is correctly folded and is transported through the secretory pathway to the cell surface. Importantly, the misfolding of the temperature-sensitive VSV G protein is reversible; thus when cells synthesizing mutant VSV G protein are grown at 40 °C and then shifted to 32 °C, the misfolded mutant VSV G protein that had accumulated in the ER will refold and be transported normally. 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 came to the same conclusion as Palade’s early experiments: 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 the 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. The hybrid gene is transfected into cultured cells by techniques. 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 the 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 complex, which are densely concentrated at the edge of the nucleus, and then to the cell surface (Figure 1a). By observing 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 1b).
Fig1. Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged membrane protein. Cultured cells were transfected with a hybrid gene encoding the viral membrane glycoprotein VSV G linked to the gene for green fluorescent protein (GFP). A temperature-sensitive mutant version of the viral gene was used so that newly made hybrid protein (VSVG-GFP) was retained in the ER at 40 °C, but was released for transport at 32 °C. (a) Fluorescence micrographs of cells just before and at two times after they were shifted to the lower temperature. Movement of VSVG-GFP from the ER to the Golgi and finally to the cell surface occurred within 180 minutes. The scale bar is 5 μm. (b) Plot of the amount of VSVG-GFP in the endoplasmic reticulum (ER), Golgi, and plasma membrane (PM) at different times after the shift to the permissive temperature. The kinetics of transport from one organelle to another can be recon structed from computer analysis of these data. The decrease in total fluorescence that occurs at later times probably results from slow inactivation of GFP fluorescence. [Jennifer Lippincott-Schwartz and Koret Hirschberg, Metabolism Branch, National Institute of Child Health and Human Development.]
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 are carrying one or more copies of the N-linked oligosaccharide Man8(GlcNAc)2, which are synthesized and attached to secretory proteins in the ER (see Figure 13-18). As a protein moves through the Golgi complex, different enzymes localized to the cis-, medial-, and trans-Golgi cisternae catalyze an ordered series of modifications to these core Man8(GlcNAc)2 chains, as dis cussed in a later section of this chapter. For instance, glycosidases that reside specifically in the cis-Golgi compartment sequentially trim mannose residues off the core oligosaccharide to yield a “trimmed” form, Man5(GlcNAc)2. 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 digestion by this enzyme (Figure 2a). Because a deglycosylated protein produced by endoglycosidase D digestion moves faster on an SDS gel than the corresponding glycosylated protein, these proteins can be readily distinguished (Figure 2b).
Fig2. Transport of a membrane glycoprotein from the ER to the Golgi can be assayed based on sensitivity to cleavage by endoglycosidase D. Cells expressing a temperature-sensitive VSV G protein were labeled with a pulse of radioactive amino acids at the nonpermissive temperature so that the labeled protein was retained in the ER. At periodic times after a return to the permissive temperature of 32 °C, VSV G protein was extracted from cells and digested with endoglycosidase D. (a) As proteins move to the cis-Golgi from the ER, the core oligosaccharide Man8(GlcNAc)2 is trimmed to Man5(GlcNAc)2 by enzymes that reside in the cis-Golgi compartment. Endoglycosidase D cleaves the oligosaccharide chains from proteins processed in the cis-Golgi, but not from proteins in the ER. (b) SDS-polyacrylamide gel electrophoresis of the digestion mixtures resolves the resistant, uncleaved (slower-migrating) and sensitive, cleaved (faster-migrating) forms of labeled VSV G protein. Initially, as this gel shows, all of the VSV G protein was resistant to digestion, but over time, an increasing fraction was sensitive to digestion, reflecting transport of the protein from the ER to the Golgi and its processing there. In control cells kept at 40 °C, only slow-moving, digestion-resistant VSV G protein was detected after 60 minutes (not shown). (c) A plot of the percentage of VSV G protein that is sensitive to digestion, derived from electrophoretic data, reveals the time course of ER-to-Golgi transport. [Part (b) republished with permission of Elsevier, from Becckers, C. J., et al., “Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex,” 1987, Cell 50(4):523–34; permission conveyed through the Copyright Clearance Center.]
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 labeled VSV G protein is still in the ER and, upon extraction, is resistant to digestion by endoglycosidase D, but over time, the fraction of the extracted glycoprotein that is sensitive to digestion increases. This conversion of VSV G protein from an endoglycosidase 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 either by the assay based on oligosaccharide processing or by fluorescence microscopy of VSVG-GFP (Figure 2c). A variety of assays based on specific carbohydrate modifications that occur in later Golgi compartments have been developed to measure progression of VSV G protein through each stage of the Golgi complex.
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. For yeast cells, as for all cells, the secretory pathway is essential for transport and delivery of new protein and membrane to the cell surface. Thus genes encoding important components of the secretory pathway are essential for cell growth and can be studied only as conditional mutants, as described in Chapter 8. Although yeasts secrete few proteins into the growth medium, they continuously secrete a number of enzymes that remain localized in the narrow space between the plasma membrane and the cell wall. The best studied of these, invertase, hydrolyzes the disaccharide sucrose to glucose and fructose.
A large number of yeast mutants were initially identified by their ability to secrete proteins at one temperature and their 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 secretory proteins at the point in the secretory 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 3). Subsequent characterization of sec mutants in these various classes has helped elucidate the fundamental components and molecular mechanisms of vesicle trafficking that we discuss in later sections.
Fig3. Phenotypes of yeast sec mutants identified five stages in the secretory pathway. These temperature-sensitive mutants can be grouped into five classes based on the site where newly made secretory 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.
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. Because 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 to the rough ER, to ER-to-Golgi transport vesicles, to Golgi cisternae, to secretory vesicles, and finally is exocytosed.
The three methods outlined in this section have delineated the major steps of the secretory pathway and have contributed to the identification of many of the proteins responsible for vesicle budding and fusion. Each of the in dividual steps in the secretory pathway is currently being studied in mechanistic detail, and increasingly, biochemical assays and molecular genetic studies are being used to study each of these steps in terms of the function of individual protein molecules.
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 mutants 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 N-linked oligosaccharide chains in the Golgi are infected with vesicular stomatitis virus, and the fate of the VSV G protein is followed. For example, if infected cells lack N-acetylglucosamine transferase I, they produce abundant amounts of VSV G protein but cannot add N-acetylglucosamine residues to the oligosaccharide chains in the medial-Golgi as wild-type cells do (Figure 4a). When Golgi membranes isolated from such mutant cells are mixed with Golgi membranes from wild-type, uninfected cells, the addition of N-acetylglucosamine to VSV G protein is restored (Figure 4b). This modification is the consequence of vesicular transport of N-acetylglucosamine transferase I from the wild-type medial-Golgi to the cis-Golgi isolated from virally infected mutant cells. Successful inter compartmental 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.
Fig4. A cell-free assay demonstrates protein transport from one Golgi cisterna to another. (a) A mutant line of cultured fibroblasts is essential in this type of assay. In this example, the cells lack the enzyme N-acetylglucosamine transferase I. In wild-type cells, this enzyme is localized to the medial-Golgi and modifies N-linked oligosaccharides by the addition of one N-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 transport vesicles from the wild-type medial-Golgi cisternae to the mutant cis-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.
In addition, under appropriate conditions, a uniform population of the transport vesicles that move N-acetylglucosamine transferase I from the medial- to cis-Golgi can be separated 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 membranes. In vitro assays similar in general design to the one shown in Figure 4 have been used to study various transport steps in the secretory pathway.
