As is evident from the preceding sections of this chapter, transport vesicles play a central role in the traffic of molecules between different membrane-enclosed compartments of the secretory pathway. As discussed in Chapter 12, vesicles are similarly involved in the transport of materials taken up at the cell surface. Vesicular transport is thus a major cellular activity, responsible for molecular traffic between a variety of specific membrane-enclosed compartments. The selectivity of such transport is therefore key to maintaining the functional organization of the cell. For example, lysosomal enzymes must be transported specifically from the Golgi apparatus to lysosomes—not to the plasma membrane or to the ER. Some of the signals that target proteins to specific organelles, such as lysosomes, were discussed earlier in this chapter. These proteins are transported within vesicles, so the specificity of transport is based on the selective packaging of the intended cargo into vesicles that recognize and fuse only with the appropriate target membrane. Because of the central importance of vesicular transport to the organization of eukaryotic cells, understanding the molecular mechanisms that control vesicle packaging, budding, and fusion is a major area of research in cell biology.

Experimental Approaches to Understanding Vesicular Transport

Progress toward elucidating the molecular mechanisms of vesicular transport has been made by three distinct experimental approaches: (1) isolation of yeast mutants that are defective in protein transport and sorting; (2) reconstitution of vesicular transport in cell-free systems; and (3) biochemical analysis of synaptic vesicles, which are responsible for the regulated secretion of neurotransmitters by neurons. Each of these experimental systems has distinct advantages for understanding particular aspects of the transport process. Most important, however, is the fact that results from all three of these avenues of investigation have converged, indicating that similar molecular mechanisms regulate secretion in cells as different as yeasts and mammalian neurons.

As in other areas of cell biology, yeasts have proved to be advantageous in studying the secretory pathway because they are readily amenable to genetic analysis. In particular, Randy Schekman and his colleagues have pioneered the isolation of yeast mutants defective in vesicular transport. These include mutants that are defective at various stages of protein secretion (sec mutants), mutants that are unable to transport proteins to the vacuole, and mutants that are unable to retain resident ER proteins. The isolation of such mutants in yeasts led directly to the molecular cloning and analysis of the corresponding genes, thereby identifying a number of proteins involved in various steps of the secretory pathway. For example, the role of Sec61 as a major component of the protein translocation channel in the endoplasmic reticulum was discussed earlier in this chapter.

Biochemical studies of vesicular transport using reconstituted systems have complemented these genetic studies and have enabled the direct isolation of transport proteins from mammalian cells. The first cell-free transport system was developed by James Rothman and colleagues, who analyzed protein transport between compartments of the Golgi apparatus (Figure 9.30). The experimental design exploited a mutant mammalian cell line that lacked the enzyme required to transfer N-acetylglucosamine residues to the N-linked oligosaccharide at an early stage of its modification in the Golgi apparatus (see Figure 9.24). Consequently, the glycoproteins produced by this mutant cell line lacked added N-acetylglucosamine units. However, if Golgi stacks isolated from the mutant cell line were incubated with stacks isolated from normal cells, N-acetylglucosamine residues were added to glycoproteins synthesized by the mutant cells. A variety of experiments established that this resulted from vesicular transport of proteins from the Golgi stacks of the mutant cell line to the Golgi stacks of normal cells, so the addition of N-acetylglucosamine provided a readily detectable marker for vesicular transport in this reconstituted system. Similar reconstituted systems have been developed to analyze transport between other compartments, including transport from the ER to the Golgi and transport from the Golgi to secretory vesicles, vacuoles, and the plasma membrane. The development of these in vitro systems has enabled biochemical studies of the transport process and functional analysis of proteins identified by mutations in yeasts, as well as direct isolation of some of the proteins involved in vesicle budding and fusion.

Figure 9.30

Reconstituted vesicular transport. Golgi stacks prepared from a virus-infected mutant cell line unable to catalyze the addition of N-acetylglucosamine to N-linked oligosaccharides are mixed with Golgi stacks from a normal cell line. Because the mutant (more...)

Critical insights into the molecular mechanisms of vesicular transport have also come from studies of synaptic transmission in neurons, which represents a highly specialized form of regulated secretion. A synapse is the junction of a neuron with another cell, which may be either another neuron or an effector, such as a muscle cell. Information is transmitted across the synapse by chemical neurotransmitters, such as acetylcholine, which are stored in synaptic vesicles. Stimulation of the transmitting neuron triggers the fusion of synaptic vesicles with the plasma membrane, causing neurotransmitters to be released and stimulating the postsynaptic neuron or effector cell. Synaptic vesicles are extremely abundant in the brain, allowing them to be purified in large amounts for biochemical analysis. Some of the proteins isolated from synaptic vesicles are closely related to proteins that have been shown to play critical roles in vesicular transport by yeast genetics and reconstitution experiments, so biochemical analysis of these proteins have provided important insights into the molecular mechanisms of vesicle fusion.

Coat Proteins and Vesicle Budding

The first step in vesicular transport is the formation of a vesicle by budding from the membrane. The cytoplasmic surfaces of transport vesicles are coated with proteins, and it appears to be the assembly of these protein coats that drives vesicle budding by distorting membrane conformation. Three kinds of coated vesicles, which appear to function in different types of vesicular transport, have been characterized. The first to be described were the clathrin-coated vesicles, which are responsible for the uptake of extracellular molecules from the plasma membrane by endocytosis (see Chapter 12) as well as the transport of molecules from the trans Golgi network to lysosomes. Two other types of coated vesicles have been identified as budding from the ER and Golgi complex. These vesicles are called nonclathrin-coated or COP-coated vesicles (COP stands for coat protein). One class of these vesicles (COPII-coated vesicles) bud from the ER and carry their cargo forward along the secretory pathway, to the Golgi apparatus. In contrast, COPI-coated vesicles bud from the ER-Golgi intermediate compartment or the Golgi apparatus and function in the retrieval pathways that serve to retain resident proteins in the Golgi and ER. For example, COPI-coated vesicles transport resident ER proteins marked by the KDEL or KKXX retrieval signals back to the ER from the ER-Golgi intermediate compartment or the cis Golgi network.

The coats of clathrin-coated vesicles are composed of two types of protein complexes, clathrin and adaptor proteins, which assemble on the cytosolic side of membranes (Figure 9.31). Clathrin plays a structural role by assembling into a basketlike lattice structure that distorts the membrane and drives vesicle budding. The binding of clathrin to membranes is mediated by a second class of proteins, called adaptor proteins. Different adaptor proteins are responsible for the assembly of clathrin-coated vesicles at the plasma membrane and at the trans Golgi network, and it is the adaptor proteins that are involved in selecting the specific molecules to be incorporated into the vesicles. For example, the AP-1 adaptor protein involved in budding from the trans Golgi network binds to the cytosolic portion of the mannose-6-phosphate receptor, thereby directing proteins destined for lysosomes into clathrin-coated vesicles.

Figure 9.31

Incorporation of lysosomal proteins into clathrin-coated vesicles. Proteins targeted for lysosomes are marked by mannose-6-phosphates, which bind to mannose-6-phosphate receptors in the trans Golgi network. The mannose-6-phosphate receptors span the Golgi (more...)

The coats of COPI- and COPII-coated vesicles are composed of distinct protein complexes, which function analogously to clathrin and adaptor proteins in vesicle budding. Interestingly, components of the COPI coat interact with the KKXX motif responsible for the retrieval of ER proteins from the Golgi apparatus, consistent with a role for COPI-coated vesicles in recycling from the Golgi to the ER.

The assembly of vesicle coats also requires GTP-binding proteins, which appear to regulate the binding of coat proteins to the membrane. The budding of both clathrin-coated and COPI-coated vesicles from the Golgi complex requires a GTP-binding protein called ARF (ADP-ribosylation factor), while the budding of COPII-coated vesicles from the ER requires a distinct GTP-binding protein called Sar1. The role of these proteins is illustrated by the function of ARF in assembly of COPI-coated vesicles (Figure 9.32). The first step in vesicle formation is the association of ARF bound to GDP with the Golgi membrane. Proteins in the Golgi membrane then stimulate the exchange of the GDP bound to ARF for GTP, and the COPI coat proteins bind to the ARF/GTP complex. Assembly of the coat is then followed by deformation of the membrane and vesicle budding. ARF then hydrolyzes its bound GTP, leading to the conversion of ARF to the GDP-bound state and the dissociation of coat proteins from the vesicle membrane.

Figure 9.32

Role of ARF in the formation of COP-coated vesicles. ARF alternates between GTP-bound and GDP-bound states. When bound to GDP, ARF associates with the membrane of the trans Golgi network, where guanine nucleotide exchange factors (GEF) promote the exchange (more...)

Vesicle Fusion

The fusion of a transport vesicle with its target involves two types of events. First, the transport vesicle must specifically recognize the correct target membrane; for example, a vesicle carrying lysosomal enzymes has to deliver its cargo only to lysosomes. Second, the vesicle and target membranes must fuse, thereby delivering the contents of the vesicle to the target organelle. Research over the last several years has led to development of a model of vesicle fusion in which specific recognition between a vesicle and its target is mediated by interactions between unique pairs of transmembrane proteins, followed by fusion between the phospholipid bilayers of the vesicle and target membranes.

Proteins involved in vesicle fusion were initially identified in James Rothman’s laboratory by biochemical analysis of reconstituted vesicular transport systems from mammalian cells (see Figure 9.30). Analysis of the proteins involved in vesicle fusion in these systems led Rothman and his colleagues to propose a general model, called the SNARE hypothesis, in which vesicle fusion is mediated by interactions between specific pairs of proteins, called SNAREs, on the vesicle and target membranes (v-SNAREs and t-SNAREs, respectively) (Figure 9.33). This hypothesis was supported by the identification of SNAREs that were present on synaptic vesicles and by the finding of yeast secretion mutants that appeared to encode SNAREs required for a variety of vesicle transport events. For example, transport from the ER to the Golgi in yeast requires specific SNAREs that are located on both the vesicle and target membranes. The formation of complexes between v-SNAREs on the vesicle and t-SNAREs on the target membranes then leads to membrane fusion, by mechanisms which remain to be fully understood.

Figure 9.33

Vesicle fusion. Vesicle fusion is mediated by binding between specific pairs of v-SNAREs and t-SNAREs on the vesicle and target membranes, respectively. Rab GTP-binding proteins are required to facilitate formation of v-SNARE/t-SNARE complexes. Following (more...)

In addition to SNAREs, vesicle fusion requires at least two other types of proteins. The Rab proteins are a family of small GTP-binding proteins that are related to the Ras proteins, which were discussed in Chapter 7. More than 30 different Rab proteins have been identified and shown to function in specific vesicle transport processes. They may function in several steps of vesicle trafficking, including interacting with SNAREs to regulate and facilitate the formation of v-SNARE/t-SNARE complexes.

Following the formation of complexes between complementary SNAREs and membrane fusion, a complex of two additional proteins (the NSF/ SNAP complex) is needed to complete the process of vesicle transport. The NSF/SNAP proteins are recruited to membranes following the formation of v-SNARE/t-SNARE complexes, and they are not required directly for either vesicle/target pairing or for the fusion of paired membranes. Instead, the NSF/SNAP proteins act after membrane fusion to disassemble the SNARE complex, thereby allowing the SNAREs to be reutilized for subsequent rounds of vesicle transport.