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Lecture 5-2012.Pdf Lecture 5 Intracellular Vesicular Transport Chapter 13 MBoC (5th Edition) Alberts et al. 2/15/12 Copyright © Garland Science1 2008 A Simplified “Roadmap” of Protein Traffic Proteins can move from one compartment to another by gated transport (red), transmembrane transport (blue), or vesicular transport (green). The sorting signals that direct a given protein’s movement through the system, and thereby determine its eventual location in the cell, are contained in each protein’s amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol and terminates when the protein reaches its final destination. At each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. In principle, a sorting signal could be required for either retention in or exit from a compartment. Figure 12-6 2 Intracellular Vesicular Traffic Exocytosis and Endocytosis Figure 13-1 (A) In exocytosis, a transport vesicle fuses with the plasma membrane. Its content is released into the extracellular space, while the vesicle membrane (red) becomes continuous with the plasma membrane. (B) In endocytosis, a plasma membrane patch (red) is internalized forming a transport vesicle. Its content derives from the extracellular space. 3 Vesicular Transport Figure 13-2 Transport vesicles bud off from one compartment and fuse with another. As they do so, they carry material as cargo from the lumen (the space within a membrane-enclosed compartment) and membrane of the donor compartment to the lumen and membrane of the target compartment. The processes of vesicle budding and fusion are not symmetrical: budding requires a membrane fusion event initiated from the lumenal side of the membrane, while vesicle fusion requires a membrane fusion event initiated from the cytoplasmic side of both donor and target membranes. 4 The Intracellular Compartments of the Eucaryotic Cell Involved in the Biosynthetic-Secretory and Endocytic Pathways Figure 13-3 *Retrieval pathways are shown with blue arrows. In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesicles. 5 Different Coats Are Utilized in Vesicular Transport Three well-characterized types of coated vesicles: each type is used for different transport steps. Figure 13-4 Different coat proteins select different cargo and shape the transport vesicles that mediate the various steps in the biosynthetic– secretory and endocytic pathways. Figure 13-5 6 The Assembly and Disassembly of a Clathrin Coat Figure 13-8 The assembly of the coat introduces curvature into the membrane, which leads to the formation of uniformly sized coated buds. The adaptor proteins bind clathrin triskelions and membrane-bound cargo receptors, thereby mediating the selective recruitment of both membrane and cargo molecules into the vesicle. The clathrin coat is rapidly lost shortly after the vesicle forms. 7 The Structure of Clathrin Coat EM of clathrin triskelion with platinum. Each triskelion is composed of three clathrin heavey chains and three clathrin light chains. Figure 13-6 EM shows clathrin-coated pits Figure 13-7 and veiscles on the inner surface A cryo-EM taken of a clathrin coat of the plasma membrane of composed of 36 triskelions organized in a cultured fibroblasts. network of 12 pentagons and 6 hexagons, with heavy chains (left) and light chains (right) highlighted. 8 The Role of Dynamin in Pinching off Clathrin-Coated Vesicles The dynamin assembles into a ring around the neck of the forming bud. The dynamin ring is thought to recruit other proteins to the vesicle neck, which, together with dynamin, destabilize the interacting lipid bilayers so that the noncytoplasmic leaflets flow together. The newly formed vesicle then pinches off from the membrane. Specific mutations in dynamin can either enhance or block the pinching- off process. A thin-section EM: Deeply invaginated clathrin-coated pits form in the fly’s nerve cells, with a ring of mutant dynamin assembled around the neck. The pinching-off process fails because membrane fusion does not take place. Figure 13-12 9 Rab Proteins Guide Vesicle Targeting Vesicle targeting depends on two types of proteins: - Rab proteins (GTPases) direct the vesicle to specific spots on the correct target membrane by interacting with Rab effectors; - SNARE proteins mediate the fusion of the lipid bilayers. Rab effector proteins interact via active Rab proteins (Rab-GTPs, yellow) located on the target membrane, vesicle membrane, or both, to establish the first connection between the two membranes that are going to fuse. In the example shown here, the Rab effector is a filamentous tethering protein (green). Next, SNARE proteins (v- and t-SNAREs) on the two membranes (red and blue) pair to dock the vesicle to the target membrane and catalyze the fusion of the two apposed lipid bilayers. Figure 13-14 10 Interacting SNAREs Need to Be Separated Before They Can Function Again Figure 13-18 Dissociation of SNARE pairs by NSF after a membrane fusion cycle. After a v-SNARE and t-SNARE have mediated the fusion of a transport vesicle on a target membrane, the NSF (ATPase) binds to the SNARE complex and, with the help of two accessory proteins, hydrolyzes ATP to pry the SNAREs apart. 11 Rab Proteins Help Ensure the Specificity of Vesicle Docking Rabs: GTPases. Each Rab has a characteristic distribution on cell membranes and every organelle has at least one Rab protein on its cytosolic surface. Proteins (downstream effectors) that are recruited or activated by Rabs include: - tethering proteins such as long fibrous proteins - large multiprotein complexes Tethering proteins link vesicles to membrane compartments and compartments to each other. 12 Transport from the ER to the Golgi Apparatus 13 The Recruitment of Cargo Molecules into ER Transport Vesicles By binding directly or indirectly to the COPII coat, membrane and soluble cargo proteins, respectively, become concentrated in the transport vesicles as they leave the ER. Figure 13-20 Membrane proteins are packaged into budding transport vesicles through interactions of exit signals on their cytosolic tails with the COPII coat. Some of the membrane proteins that the coat traps function as cargo receptors, binding soluble proteins in the lumen and helping to package them into vesicles. A typical 50-nm transport vesicle contains about 200 membrane proteins, which can be of many different types. As indicated, unfolded or incompletely assembled proteins are bound to chaperones and retained in the ER compartment. 14 A Model for the Formation of a COPII-Coated Vesicle A complex of two additional Sar1: a coat COPII coat proteins, called recruitment Sec13/31, forms the outer GTPase shell of the coat. Membrane bound, active Sar1-GTP recruits COPII subunits to the membrane, causing the membrane to form a bud, which includes selected transmembrane proteins. A subsequent membrane-fusion event pinches off the coated vesicle. GTP-bound Sar1 binds to a complex of two COPII coat proteins, called Sec23/24. 15 Figure 13-13 Vesicular Tubular Clusters (B) Figure 13-23 (A) (A) An EM section of vesicular tubular clusters forming from the ER membrane. Many of the vesicle-like structures seen in the micrograph are cross sections of tubules that extend above and below the plane of this thin section and are interconnected. (B) Vesicular tubular clusters move along microtubules to carry proteins from the ER to the Golgi apparatus. COPI coats mediate the budding of vesicles that return to the ER from these clusters. 16 A Model for the Retrieval of Soluble ER Resident Proteins Figure 13-24 (A) (B) ER resident proteins that escape from the ER are returned by vesicular transport. (A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER. Upon binding its ligands in this environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPI-coated vesicles. (B) The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus. In the environment of the ER, the ER resident proteins dissociate from the KDEL receptor, which is then returned to the Golgi apparatus for reuse. 17 The Golgi Apparatus Consists of an Ordered Series of Compartments (A) 3-D reconstruction from EMs of the Golgi apparatus in a secretory animal cell. The cis face of the Golgi stack is that closest to the ER. (B) A thin section EM emphasizing the transitional zone between the ER and the Golgi apparatus in an animal cell. (C) An EM of a Golgi apparatus in a plant cell (the green alga Chlamydomonas) seen in cross section. In plant cells, the Golgi apparatus is generally more distinct and more clearly separated from other intracellular membranes than in animal cells. (A) (C) 18 (B) Figure 13-25 The Functional Compartmentalization of the Golgi Apparatus Oligosaccharide processing in Golgi compartments. Processing enzymes are not restricted to a particular cisterna; instead, their distribution is graded across the stack— such that early-acting enzymes are present mostly in the cis Golgi cisternae and later-acting enzymes are mostly in the trans Golgi cisternae. Figure 13-28 19 Oligosaccharide Processing in the ER and the Golgi Apparatus Figure 13-31 Endo H: a highly specific endoglycosidase. Two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian glycoproteins. Sometimes, both types are attached, in different places, to the same polypeptide. The oligosaccharide processing pathway is highly ordered, so that each step shown depends on the previous one. 20 Models for Transport Through the Golgi Apparatus • Individual proteins and small protein structures are transported through the Golgi apparatus either by cisternal maturation or vesicle-mediated transport.
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