Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Intracellular Macromolecular Sorting and Maintenance of Cellular Compartments
Vesicular Transport and the Maintenance of Compartment Identity

Intracellular sorting evidently requires at least 10 Different types of transport vesicles, each bearing a unique set of "molecular address tags" on its surface that ensure cargo delivery exclusively to the appropriate target membrane. Consequently, transport vesicles budding from the ER must fuse solely with the cis-Golgi network, those leaving the cis-compartment must fuse only with the medial Golgi compartment, and so forth. Each stage involves vesicle budding, docking, and membrane fusion—processes that demand a high degree of specific recognition. Currently, our understanding of these molecular mechanisms remains limited (see Fig. 8-73). In this section, we examine some competing hypotheses regarding how intracellular compartmentalization is maintained and introduce novel experimental approaches designed to resolve these debated issues.

8.10.1. Certain Proteins Are Retained as Permanent Residents of the ER and Golgi apparatus [67]

As cargo moves from one compartment to another, transport vesicles necessarily carry both membranes and organelle contents. Despite this ongoing redistribution, the distinct membrane compositions of different compartments are remarkably well-maintained: the SRP receptor protein is found exclusively in the ER membrane, whereas Glycosyltransferases and oligosaccharide-Processing Enzymes reside solely within the membranes of specific Golgi cisternae, and so on. Consequently, the membranes of the ER and each type of Golgi cisterna must possess specialized mechanisms to preserve their unique identities. One such mechanism is the presence of specific sorting signals that direct proteins at each step of their transit through the ER and Golgi apparatus. For instance, plasma Membrane Proteins entering The Cell via receptor-mediated endocytosis are selectively captured by coated pits. However, an alternative view suggests that biosynthetic transport through the ER and Golgi operates via the opposite mechanism—meaning that forward transport occurs by default, and specific signals are required strictly for protein retention within an organelle. According to this hypothesis, every resident protein of the ER or Golgi apparatus must possess a distinct retention signal that prevents its escape. This strategy of automatic forward movement coupled with selective retention is appealing because the number of proteins trafficking through the ER and Golgi to their final destinations vastly exceeds the number of resident proteins that stay behind. Furthermore, this strategy provides a built-in quality control mechanism: proteins that lose their sorting signals or are misrouted can be efficiently cleared from the cell. Finally, if transport signals were required for forward movement, they would be needed at every single step—from the ER to the Golgi, and from each Golgi cisterna to the next. Such a requirement would place a heavy structural burden on numerous proteins, forcing them to accommodate multiple surface sorting signals.

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8.10.2. At Least Two Types of Coated Vesicles Exist [68]

Clathrin is localized to the cytoplasmic surface of The Plasma Membrane and the trans-Golgi network, where it appears to mediate signal-directed transport. Clathrin-coated vesicles drive both the receptor-mediated endocytic pathway from the plasma membrane to endosomes and the receptor-regulated pathway from the trans-Golgi network to endolysosomes. In the former case, these coated vesicles transport a carefully selected subset of cell-surface receptors. This principle likely applies to other classes of clathrin-coated vesicles budding from the Golgi apparatus as well.

Clathrin-coated vesicles do not bud from the ER or from the cis- and medial cisternae of the Golgi apparatus. Instead, these regions (as well as the trans-Golgi network) utilize a different Class of coated vesicles. These structures do not stain with Antibodies against clathrin and appear distinctly different from clathrin-coated vesicles under the Electron microscope (Fig. 8-83). The coat proteins of these vesicles have not yet been identified.

A putative model of protein transport in unpolarized Cells is illustrated in Fig. 8-84. According to this model, proteins move "by default" from the ER to the Golgi apparatus, from one Golgi cisterna to the next, and ultimately from the trans-Golgi network to the cell surface. This non-selective bulk flow is thought to be mediated by non-clathrin-coated vesicles. From the trans-Golgi network, receptor-mediated transport to Lysosomes relies on clathrin-coated vesicles, whereas trafficking to secretory granules involves partially clathrin-coated vesicles. How might this model be tested experimentally?

Fig. 8-83. Comparison of clathrin-coated and non-clathrin-coated vesicles. (A) Electron micrograph of Golgi cisternae from a cell-free system undergoing the in vitro budding of non-clathrin-coated vesicles. The clathrin-coated vesicles shown in (B) exhibit a much more regular, lattice-like Structure. (Electron micrographs kindly provided by Lelio Orci; see L. Orci, B. Glick, and J. Rothman, Cell, 46: 171–184.)

Fig. 8-84. Hypothetical model of protein transport in unpolarized cells. Non-selective (constitutive) transport is proposed to occur via non-clathrin-coated vesicles, whereas Various Forms of signal-mediated transport utilize clathrin-coated vesicles. The latter bud from the trans-Golgi network, endosomes, and the plasma membrane. Polarized cells require an additional layer of signal-regulated transport originating from the trans-Golgi network.

8.10.3. Secretory Mutant Cells Help Unravel the Transport Mechanism [69]

Because Intracellular Transport is vital for Introduction/5.html">Eukaryotic Cell survival, mutant cells defective in any essential step of this pathway (such as Mutations affecting a docking marker or a receptor protein) would normally be lethal. However, if a mutant protein becomes non-functional only at elevated temperatures, the cell can remain perfectly viable under normal conditions. Genetic screens in Yeast have identified over 25 Temperature-sensitive mutations in genes required for secretion. Cells carrying these mutations fail—at restrictive temperatures—to transport proteins from the ER to the Golgi, between Golgi cisternae, or from the Golgi to the vacuole or plasma membrane.

Taking advantage of these temperature-sensitive yeast mutants makes it straightforward to clone the corresponding transport genes. This approach has proven exceptionally fruitful because it allows the direct identification of Key Components of the transport machinery without requiring prior knowledge of how the secretory process works. One such yeast secretory Gene identified through this approach is sec4, which is believed to encode a ras-family GTP-binding protein (see Sections 12.3.11 and 13.4.6). Biochemical experiments in mammalian cells indicate that a homologous GTP-binding protein participates in Vesicular Transport in higher eukaryotes as well, potentially regulating the "uncoating" of non-clathrin-coated vesicles prior to their membrane binding.

8.10.4. Cell-Free Systems Provide Another Powerful Approach for Studying the MOLECULAR MECHANISMS OF Vesicular Transport [70]

To understand the molecular mechanisms underlying the flow of molecules between membrane compartments, researchers must isolate the core "machinery" driving transport vesicles. How do transport vesicles bud from a membrane? What guides them to their target membranes? How do they fuse with those targets? In addition to the genetic approaches described above, reconstitution assays of vesicular transport in cell-free (in vitro) systems have been instrumental in answering these questions. This was first successfully achieved using isolated Golgi stacks. When isolated Golgi stacks were incubated with [³H]-GlcNAc derived from UDP-[³H]-GlcNAc added to the culture medium, transport occurred—as shown in the graph on the right—only when both cytosolic extracts and ATP were supplied. These exact same conditions are required for the budding of non-clathrin-coated vesicles. This experimental design, initially developed to study protein transfer from the cis-Golgi to the medial Golgi compartment, has since been successfully adapted to reproduce in vitro Transport from the ER to the cis-Golgi, from medial to trans-Golgi compartments, from the trans-Golgi network to the plasma membrane, from endosomes to lysosomes, and from the trans-Golgi network to endolysosomes.

Fig. 8-85. A cell-free system reconstituting the vesicular transport of a viral protein between Golgi cisternae. When preparations of isolated Golgi stacks are incubated with cytosolic extracts and ATP, non-clathrin-coated vesicles bud off from their rims. Vesicular stomatitis virus (VSV) encodes a coat glycoprotein known as the G protein. To determine how this transmembrane protein is transported between Golgi cisternae, two distinct populations of isolated Golgi stacks were incubated together. The "donor" population was harvested from VSV-infected mutant cells lacking GlcNAc transferase I (meaning that the donor Golgi stacks cannot incorporate GlcNAc residues into the N-linked Oligosaccharides of the G protein). The "acceptor" Golgi stacks were isolated from uninfected wild-type cells and thus contained normal GlcNAc transferase activity but lacked the G protein. Consequently, for the G protein to acquire GlcNAc, it had to travel via transport vesicles from the cis-compartment of the donor Golgi stack to the medial compartment of the acceptor stack. Transport-coupled glycosylation can thus be monitored by measuring the incorporation of [³H]-GlcNAc into the G protein.

supplemented with Cytosol and ATP, non-clathrin-coated vesicles pinched off from their margins, apparently carrying proteins between cisternae (Fig. 8-83A). By tracking the sequential processing of oligosaccharides on Glycoproteins as they advanced from one Golgi compartment to the next, researchers successfully reconstructed vesicular transport in vitro (Fig. 8-85).

Experiments revealed that the budding of non-clathrin-coated vesicles in this cell-free system requires a combination of cytosolic proteins and ATP, indicating that it is an active, energy-consuming process rather than simple self-assembly. The molecular components essential for transport are remarkably conserved evolutionarily; animal cell cytosol can be successfully replaced in these reconstitution assays by cytosol extracted from yeast or plants. Thus, combining genetic approaches in yeast with mammalian cell-free systems offers a powerful synergistic strategy for identifying the molecules involved in vesicular transport.

Summary

The ER and each compartment of the Golgi apparatus contain unique sets of resident proteins. These proteins are apparently retained within their respective Organelles through the action of specialized targeting signals. Meanwhile, the bulk of cellular cargo moves automatically from the ER to the Golgi, through the Golgi stack, and from the trans-Golgi network to the cell surface. This non-selective bulk flow is thought to be mediated by non-clathrin-coated vesicles that do not sort cargo based on specific signals. In contrast, signal-directed transport (sorting) is mediated by clathrin-coated vesicles. Testing these hypotheses requires deciphering the molecular machinery that drives vesicle budding, targeting, and fusion. Genetic screens in yeast have identified more than 25 genes whose products govern distinct stages of transport. Furthermore, cell-free systems developed for mammalian cells have successfully recapitulated selective vesicle budding and fusion. Ultimately, the integration of genetic and biochemical approaches promises to isolate and characterize the many proteins orchestrating these fundamental cellular processes.

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