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Fundamentals of Cell Biology Chapter 9: The Endomembrane System and Membrane Trafficking

Chapter Summary: The Big Picture (1) Chapter foci: Define the endomembrane system and introduce the general concepts of vesicle-mediated traffic Understand the temporal sequence of events beginning with ER and ending at PM Explore details of Golgi function Understand Golgi to PM pathway The role of the endosome in sorting The significance of the lysosome

Chapter Summary: The Big Picture (2) Section topics: The endomembrane system is a network of organelles in eukaryotic cells Exocytosis begins in the endoplasmic reticulum The Golgi apparatus modifies and sorts proteins in the exocytic pathway Exocytosis ends at the plasma membrane Endocytosis begins at the plasma membrane The endosome sorts proteins in the endocytic pathway Endocytosis ends at the lysosome

The endomembrane system is a network of organelles in eukaryotic cells Key Concepts (1): The endomembrane system is a set of interconnected organelles that readily exchange materials. The primary functions of the endomembrane system are to control the export (exocytosis) and import (endocytosis) of materials to/from the extracellular space.

The endomembrane system is a network of organelles in eukaryotic cells Key Concepts (2): Membrane-bound compartments called vesicles shuttle between organelles in the endomembrane system and are responsible for carrying material from one organelle to another. The creation, transport, targeting, and fusion of vesicles occurs in nine steps.

The endomembrane system controls molecular transport in/out of cell Endocytic and exocytic pathways Endomembrane system Figure 09.01: The endocytic and exocytic pathways.

Vesicles shuttle material between organelles in the endomembrane system Donor/acceptor compartments Vesicle-mediated transport Anterograde/retrograde transport = vesicle shuttling Exocytosis, endocytosis, and fusion of vesicles Figure 09.02: In vesicle-mediated transport, a membrane-bound vesicle buds from one compartment and fuses with another.

Vesicle-mediated transport - 9 steps Figure 09.03: Vesicle-mediated transport occurs in nine steps. Cargo selection Budding Scission Uncoating Transport Tethering Docking Fusion Disassembly

Vesicle-mediated transport - 9 steps Figure 09.04: The three main types of coats involved in vesicle-mediated transport are COPI, COPII, and clathrin coats. Cargo selection Budding Scission Uncoating Transport Tethering Docking Fusion Disassembly

Mneumonic Contest C B S U T T D F D D Best mneumonic for the 9 steps wins 2 points on their choice of midterm exams (1, 2 or 3) C B S U T T D F D D

Exocytosis begins in the ER Key Concepts: Newly synthesized endomembrane proteins are modified in the ER. Signaling sequences in the newly-made proteins signal their secondary destinations. Some have retention signals that keep them in the ER. Proteins that leave the ER enter the cis-Golgi network via COPII-coated vesicles. Retrograde vesicle transport returns ER-resident proteins from the Golgi apparatus to the ER.

Job of exocytic pathway – sort/distribute new proteins in endomembrane system Newly synthesized proteins begin post-translational modification as ER resident proteins help them fold properly COPII-coated vesicles shuttle proteins from the ER to the Golgi apparatus Resident ER proteins are retrieved from the Golgi apparatus

Exocytic pathway Figure 09.05: Cargo proteins are incorporated into nascent COPII vesicles by different mechanisms. Figure 09.06: ER-resident proteins are occasionally carried to the CGN and must be retrieved.

The Golgi apparatus modifies and sorts proteins in the exocytic pathway Key Concepts (1): The Golgi apparatus is organized into discrete compartments called cisternae. The cisternae are stacked on top of one another, and are classified as cis, medial, or trans according to their relative location within the overall Golgi structure. Golgi-resident proteins are primarily responsible for modifying proteins undergoing exocytosis. They are retained in the Golgi apparatus by transmembrane Golgi retention sequences.

The Golgi apparatus modifies and sorts proteins in the exocytic pathway Key Concepts (2): The extreme ends of the Golgi apparatus are elaborated into long, tubular structures called the cis Golgi network and trans Golgi network. Both Golgi networks sort proteins into vesicles targeted to different locations. The trans Golgi network is especially effective at sorting a large number of proteins into many distinct vesicle types.

Site for post-translational modification (PTM) The Golgi apparatus is subdivided into cis, medial, and trans cisternae Site for post-translational modification (PTM) Golgi proteins retained by retention sequence Figure 09.08: Proteins leaving the TGN can travel to at least five different destination compartments.

The trans Golgi network (TGN) sorts proteins exiting the Golgi apparatus Figure 09.09: Dynamin uses GTP to regulate scission of a vesicle from a donor compartment.

TGN sorting mechanisms are complex Many different “cargo selection” mechanisms PTM Protein aggregation Signal receptor Lipid raft

Budding of vesicles at the TGN likely occurs by several different mechanisms 3 different mechanisms have been proposed Curvature-inducing proteins Modification of membrane phospholipids Phospholipid asymmetry

Scission of budding vesicles requires microtubule motor-based forces Dynamin - GTPase Figure 09.10: Examples of the golgi cisternae, rough ER (RER), and various stages of secretory granule formation. Ly = lysosome.

Exocytosis ends at the plasma membrane Key Concepts: Cells regulate the last stage of exocytosis (fusion) for most exocytic vesicles, to control when and how much material is released into the extracellular space and to control the delivery of membrane-associated proteins to the plasma membrane. Controlled secretion is also called regulated secretion, and is under the control of signaling pathways.

2 mechanisms for controlling the final steps of exocytosis Constitutive secretion constant Regulated secretion Controlled by signaling proteins Secretory vesicles (zymogen granules) Condensing vacuole Figure 09.11: Transmission electron micrograph of clathrin-coated pits and vesicles at the oocyte surface.

Exocytosis ends at the plasma membrane Key Concepts: Cells regulate the last stage of exocytosis (fusion) for most exocytic vesicles, to control when and how much material is released into the extracellular space and to control the delivery of membrane-associated proteins to the plasma membrane. Controlled secretion is also called regulated secretion, and is under the control of signaling pathways.

Endocytosis begins at the plasma membrane Key Concepts: The onset of endocytosis is most often indicated by the clustering of cargo receptors on the plasma membrane, accompanied by the assembly of a clathrin coat on the cytosolic face of the cluster. In micrographs, this structure resembles a pit in the membrane, so it is often called a coated pit. Coated pits complete the nine steps of vesicle transport and deliver the vesicle to an organelle called the endosome.

Clathrin stabilizes the formation of endocytic vesicles Figure 09.12: The role of clathrin in endocytosis. Figure 09.13: Clathrin-coated vesicles are uncoated and tethered to early endosomes via EEA1, which binds to phosphatidylinositol-3-phosphate and Rab5.

The endosome sorts proteins in the endocytic pathway Key Concepts (1): The endosome is formed by the fusion of endocytic vesicles with specific vesicles that bud from the TGN. The endosome sorts materials arriving from the plasma membrane; cargo receptors are recycled to the plasma membrane, while cargo remains in the endosome.

The endosome sorts proteins in the endocytic pathway Key Concepts (2): The lumen of the endosome is slightly acidic relative to the extracellular space, and this acidity is key to the sorting mechanism. This sorting mechanism is very different from the sorting mechanisms used in the Golgi apparatus.

The endosome is subdivided into early and late compartments Specific interacting molecules ensure correct vesicles fuse with vesicles from TGN or early endosomes EEA-1,Syntaxin,Rab 5, PIP3 Proton pump proteins play central role in sorting/activation of endosomal contents Figure 09.14: Maturation of endosomes to form lysosomes. Sorting of cell surface receptors and M6P receptors requires acidic environment provided by proton pumps.

Endocytosis ends at the lysosome Key Concepts (1): Complete degradation of endocytosed materials takes place in an organelle called the lysosome. The lysosome is likely generated from the endosome in several ways, and requires fusing a vesicle from the TGN that contains essential proton pump proteins and digestive enzymes with the endosome.

Endocytosis ends at the lysosome Key Concepts (2): The digestive enzymes and proton pumps are carried to the endosome by receptors that recognize a mannose 6-phosphate tag on proteins in the TGN. The mannose 6-phosphate receptors are sorted and returned to the TGN, while the hydrolases and pumps remain in the endosome. The pumps are responsible for triggering the drop in pH in the endosomes.

Endocytosis ends at the lysosome Key Concepts (3): Once the plasma membrane cargo receptors and mannose 6-phosphate receptors have been removed from the endosome, it becomes progressively more acidic; when it reaches pH 5, shape changes in the hydrolases are triggered, which activate the endosomes and allow them to begin digesting cargo. Once digested, the cargo building blocks (sugars, nucleosides, amino acids, etc.) are transported into the cytosol for reuse.

Maturation from endocytic vesicle to lysosome Figure 09.15: Assembly of the mannose 6-phoshate sorting signal.

Figure 09.16: Digestion of cargo molecules in the lysosome. Endogenous proteins destined for lysosome are tagged and sorted by Golgi Figure 09.16: Digestion of cargo molecules in the lysosome.

pH is used in 3 ways to control endocytosis 1) the acidic environment in endosomes helps sort cargo from receptors 2) the relatively neutral pH of the ER and Golgi apparatus keeps the hydrolytic enzymes from digesting these organelles 3) the enzymes’ requirement for a strong acid environment protects the endomembrane system from digesting itself

Digestion in the lysosome Proteases Glycosidases DNaseII Phosphatases Figure 09.17: Ubiquitin-mediated degradation occurs in multivesicular bodies, which are late endosomal compartments of the endocytic pathway.

Lysosomes can also degrade some resident organelles “Autophagy” by autophagosome