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Protein Sorting & Transport

Protein Sorting & Transport. Paths of Protein Trafficking Nuclear Protein Transport Mitochondrial & Chloroplast Transport Experimental Systems Overview of the Cytomembrane System The Endoplasmic Reticulum The Golgi Apparatus Vesicular Transport between Compartments Exocytosis

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Protein Sorting & Transport

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  1. Protein Sorting & Transport Paths of Protein Trafficking Nuclear Protein Transport Mitochondrial & Chloroplast Transport Experimental Systems Overview of the Cytomembrane System The Endoplasmic Reticulum The Golgi Apparatus Vesicular Transport between Compartments Exocytosis Endocytosis and Lysosomes

  2. Experimental Systems 1. Autoradiography, “Pulse-Chase,” and Green Fluorescent Protein Experiments 2. Differential Staining 3. Cell Fractionation 4. Genetic Mutant Analysis

  3. Nuclear Protein Transport • Nuclear Pores ~9 nm formed from nucleoporin proteins • Smaller proteins (~17kD or less) may diffuse freely; larger proteins must be imported by “gated pore” mechanism • Proteins targeted for nucleus have nuclear localization signals • Most NLS must be recognized & bound by nuclear import receptors or nuclear export receptors

  4. Nuclear Protein Transport • During nuclear protein transport, much of the tertiary structure of the transported protein remains intact, due to large size of nuclear pores • Energy is provided by GTP hydrolysis via Ran, a GTPase found both in cytoplasm & nucleus • In the cytoplasm a GTPase activating factor (GAP) triggers hydrolysis of GTP by Ran; in the nucleus a GDP-GTP exchange factor (GEF) • The gradient of Ran-GDP and Ran-GTP across the nuclear membrane drives transport in the appropriate direction

  5. Mitochondrial/Chloroplast ProteinTransport • Mitochondrial proteins that are encoded by nuclear genes possess mitochondrial import sequences • Transport from the cytosol into the mitochondria is mediated by a TOM complex and two TIM complexes • The SAM complex mediates proper folding of outer membrane proteins with β-barrel structures • Another complex (OXA) mediates insertion of mitochondrial-encoded proteins into inner membrane & also contributes to insertion of some nuclear-encoded proteins • Proteins must be unfolded, either by chaperones or by specialized unfolding proteins

  6. Mitochondrial/Chloroplast ProteinTransport • ATP hydrolysis & a H+ gradient drive protein transport into mitochondria via a ratcheting mechanism • Integral proteins possess stop transport sequences that interrupt the transport process to create the transmembrane domains; formation of the transmembrane domain may be either mediated by TIM23, TIM22 (specialized for multipass proteins), or OXA

  7. Mitochondrial/Chloroplast ProteinTransport • Transport of chloroplast proteins is similar to mitochondrial proteins • Thylakoid proteins require an extra thylakoid transport sequence & import proteins

  8. Overview of the Cytomembrane System 1. Endoplasmic Reticulum 2. Golgi Apparatus 3. Intercompartmental Transport Vesicles 4. Secretory Vesicles 5. Endocytotic Vesicles 6. Lysosomes

  9. Endoplasmic Reticulum 1. Functions of the Smooth ER • Steroid Hormone Synthesis • Detoxification in Liver • Release of glucose from liver • Sequestering calcium ions

  10. Endoplasmic Reticulum 2. Functions of the Rough ER • Cotranslational import of proteins destined for the ER-Golgi pathway • Synthesis of membrane lipids and generation of lipid compositional asymmetry • Protein glycosylation: Synthesis of the core portion of an N-linked oligosaccharide

  11. Golgi Apparatus • Modification of N-linked oligosaccharides • Synthesis of O-linked oligosaccharides • Phosphorylation of mannose (on N-linked oligosaccharide) on proteins targeted for lysosomes • Sorting of proteins into secretory vesicles or primary lysosomes

  12. Vesicular Transport between Compartments 1. Transport vesicles are generally covered with coat proteins: • COPII-coated vesicles: move proteins from ER to cis-Golgi • COPI-coated vesicles: move proteins from cis-Golgi to ER; also possibly from ER to Golgi and between Golgi cisternae • Clathrin-coated vesicles: move proteins from the trans-Golgi to the plasma membrane or lysosomes

  13. Vesicular Transport between Compartments 2. Receptor protein systems (SNAREs) are believed to target and dock specific vesicles to the correct compartment 3. At each step in the cytomembrane pathway, proteins that should stay in the previous compartment are retrieved by membrane-bound receptors and sent back to the correct compartment.

  14. Exocytosis • Secretory vesicles (from the trans-Golgi) are targeted to the plasma membrane, with which they fuse. • The soluble contents of the vesicles are released to the outside, and the vesicle membrane becomes part of the PM.

  15. Endocytosis and Lysosomes • Endocytotic vesicles form from clathrin-coated pits in the plasma membrane. This process is often mediated by receptor proteins that bind to specific ligands that the cell need to transport. • Generally, the endocytotic vesicles fuse with primary lysosomes (from the trans-Golgi) to form secondary lysosomes.

  16. Endocytosis and Lysosomes • Lysosomes contain many different hydrolytic enzymes that process the contents of the endosome. • Lysosome proteins are recognized and sorted by the trans-Golgi due to the mannose 6-phosphate residues on N-linked oligosaccharide

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