Lecture 13 Vesicular Traffic 2024 PDF

Vesicular Traffic Frog NMJ 1  Transport From the ER to the Golgi and Beyond We continue our discussion from last lecture in which proteins were translocated to the ER. Next, they are transported to the Golgi apparatus via protein-coated vesicles. We discuss these...

Vesicular Traffic Frog NMJ 1  Transport From the ER to the Golgi and Beyond We continue our discussion from last lecture in which proteins were translocated to the ER. Next, they are transported to the Golgi apparatus via protein-coated vesicles. We discuss these here. **In this lecture, we will deal primarily with the anterograde (forward) movement of proteins. 2  Types of Coated Vesicles PM-Golgi from Golgi from ER 3 Types of Coated Vesicles Arrows = Golgi cisternae 4  COPII-coated Transport Vesicles Sar1 is a coat-recruitment GTPase (mol. switch) in cytosol. GDP replaced by GTP to produce Sar1-GTP. Sar1-GTP binds to ER membrane via hydrophobic tail and initiates membrane curvature. Sar1-GTP recruits coat proteins (e.g. Sec23 and 24). Role in assembly of coated vesicles; hydrolysis reverses process. Coat proteins recruit cargo receptors. Cytosol 5  Budding of COPII Vesicles Properly-folded proteins are selectively concentrated inside vesicles. “Cargo proteins” are recognized by cargo receptors. Budding of vesicles occurs at exit sites to transport both soluble and transmembrane proteins. Outer coating proteins, e.g. Sec13/31. 6 SNARE Proteins and Target Specificity “SNAREs” are surface markers on vesicles that identify cargo type and bind to complementary receptors. v-SNAREs: vesicle membrane. t-SNAREs: target membrane. trans SNARE complexes. Membrane fusion. Rab proteins in GTP state will bind to Rab effector proteins. Figs. 13-11 Alberts, 4th ed. Soluble NSF Attachment Protein REceptor GDI = Rab-GDP dissociation inhibitor 7  Docking and SNARE Dissociation SNAREs mediate docking and membrane fusion. NSF is an ATPase that ‘pries’ SNAREs apart so they can be used again in transport process. With accessory proteins, NSF hydrolyzes ATP. 8 NSF = N-methylmaleimide-sensitive fusion protein  Homotypic Membrane Fusion Budded vesicles from ER fuse with each other. Addition of adaptor proteins, NSF, and ATP pry SNARE proteins apart. Recognition of SNAREs from different vesicles leads to membrane fusion. See previous slide 9  Vesicular Tubular Clusters Formed by fusion of COPII vesicles of ER. Function as transport packages to cis Golgi network. Travel along MTs and deliver contents. 10  Transport Through the Golgi As proteins move through the Golgi apparatus, they are modified by a series of chemical reactions. These reactions help to sort proteins so that they will be directed to their appropriate sites once they emerge. 11  A Golgi “Stack” Series of membrane-enclosed cisternae linked by tubular connections forming a single complex. 2 “faces”: cis (entry) and trans (exit). Each associated with specialized tubular network. From ER (Entry) Anterograde transport (Exit) To vesicles, membrane, 12 cell exterior etc.  Glycosylation Addition of one or more sugars to a protein (or lipid) 1. N-linked glycosylation: NH2 group, asparagine. produces 2 types of oligosaccharides: complex oligosaccharides and high-mannose oligosaccharides. 2. O-linked glycosylation: OH group, serine or threonine. Glycosylation follows specific sequence of reactions. Proteins thus directed to specific destinations. 13  Functional Compartmentalization Key for N-linked Oligosaccharides Glycosylation normally complete Segregation of glycosylated proteins e.g. removal of mannose in cis and medial cisternae is important for sorting to plasma membrane 14 2 Models for Protein Transport Between Cisternae “Dynamic” “Static” 15  Transport from the Golgi Onward Proteins emerge from the trans Golgi network and are sent to appropriate destinations. Transport vesicles carry proteins and lipids bound for the plasma membrane, and water soluble proteins for exocytosis. 2 secretory pathways: constitutive and regulated. In the regulated pathway, a signal directs secretory proteins to secretory vesicles. 16 2 Secretory Pathways e.g. neurons 17  Formation of Secretory Vesicles Increasing acidity of vesicle lumen Segregation and concentration of secretory proteins. This occurs by: 1. Progressive acidification of vesicle lumen driven by H+ ATPase activity. 2. Removal of membrane and lumen material (recycling) from vesicle by clathrin during maturation. TEM image of insulin-secreting cell of pancreas. Immature vesicle (open arrow) still contains some clathrin. Mature vesicle (closed arrow) does not. 18  Release from Secretory Vesicles Vesicles travel to plasma membrane (e.g. by axonal transport in neurons). Wait for extracellular signal (e.g. chemical or electrical signal). Increased cytosolic Ca2+ may trigger release. Contents discharged to the cell exterior. Plasma membrane SA temporarily increases. Proteins of vesicle either degraded or recycled. 19 Release from Secretory Vesicles 20 Synaptic Vesicle Formation Figure 13-74 Alberts 5th ed. 21  Synaptic Vesicle Release TEM and SEM images Pre-stimulation from frog neuromuscular junction. Stimulation causes Ca2+-dependent release of neurotransmitter (ACh). Post-stimulation 22  Things to Consider... 1. Can you describe the path of a protein from ribosome to plasma membrane? 2. Why would cells have a regulated secretory pathway in addition to a constitutive one? 23