Cell Biology Protein Translocation and Secretory Pathway PDF

XY1110 Cell Biology Lecture 3: Protein Translocation and the Secretory Pathway Kamran Manzoor [email protected] Slides by Dr Peter Oatley (poat...

XY1110 Cell Biology Lecture 3: Protein Translocation and the Secretory Pathway Kamran Manzoor [email protected] Slides by Dr Peter Oatley ([email protected]) Protein Translocation and the Secretory Pathway Lecture Structure Protein Targeting Misfolded Membrane Proteins Eukaryotic Secretory - Pathogenicity Pathway Rough/Smooth ER recap Protein Translocation Secretory Pathway - Modifications - Quality control - Misfolded Proteins Protein Translocation and the Secretory Pathway Learning objectives Know the differences between the rough and smooth ER Understand the differences between co- and post-translational translocation Explain what happens to proteins in the ER in relation to post-translational modification and quality control Understand what happens to misfolded proteins in the ER Know the cellular pathogenesis and consequence of defective protein folding. Identify and understand medical conditions associated with defective transmembrane protein folding  Protein Translocation Protein Targeting - Most proteins are made in the cytosol - For proteins to function correctly they need to be directed to the correct cellular compartment Cytosol Lectur Lecture e4 4 Nucleus Peroxisome Lectur s N/A for Medical e6 Sciences Mitochondria Plastids Mostly co-translational in humans (This lecture) Endoplasmic Reticulum Protein Translocation and the Secretory Pathway ukaryotic Secretory Pathway Protein Endoplasmic Translocatio Reticulum n Golgi Secretor Late y Endosome Vesicles Lysosom e Early Endosome Cell Exterior Molecular Biology of  Protein Translocation Eukaryotic Cell - Organelles RECAP Smooth endoplasmic reticulum - Important for lipid: - Lipid processing - Lipid secretion Molecular Biology of  Protein Translocation Eukaryotic Cell - Organelles RECAP Rough endoplasmic reticulum - ER studded with ribosomes - Protein production: - Membrane proteins - Extracellular proteins - Secretory pathway Molecular Biology of  Protein Translocation rotein Translocation - Signal Sequence Targeting signal - ER Signal Sequence - N-Terminal signal sequence - Target proteins to the ER Signal Sequence features: - Positive charge (N-region) - Hydrophobic (H-region) - Signal peptidase (C-region) Yeast Molecular Biology of  Protein Translocation otein Translocation - Co-translational translocation Co-translational translocation of Proteins in Eukaryotes - Translocation across the ER membrane during translation at rough ER ribosomes SRP binds to SRP guides the SRP and SRP the signal signal Receptor sequence, sequence to the released translation SRP Receptor Translation pauses at the ER drives translocation Signal Sequence SRP Sec SRP Complex Receptor Molecular Biology of (SR)  Protein Translocation otein Translocation - Co-translational translocation Co-translational translocation of Proteins in Eukaryotes - Translocation across the ER membrane during translation at rough ER ribosomes - Signal sequence is cleaved by signal peptidase - Ribosome components recycled Molecular Biology of  Protein Translocation otein Translocation - Post-translational translocation Post-translational translocation of Proteins - Targeting of proteins to eukaryotic organelles after complete translation Post-translational targeting to the ER in Humans - Little is know in humans - Most secretory proteins co-translationaly targeted - Much of the work has been done in yeast In yeast, requires: Sec61 Complex - Core translocon Sec62/63 Complex - Substrate binding Sec61 Complex and recognition BiP - “Binding Protein” ATPas chaperone in the ER Substrates: Secretory proteins, single Molecular pass Biology of and double pass membrane proteins  Protein Translocation Secretory Pathway - Protein Modifications Post-translational modification of proteins in the ER Glycosylati - Proteins are modified in the secretory pathway on ER Modifications - N-linked glycosylation - Protein folding with chaperones - Proteolytic cleavage - Protein oligomerisation - Disulphide bonds form (ProteinChaperone disulfide- Disulphide isomerase, PDI) s bonds Golgi Modifications - O-linked glycosylation - Modification of glycosylations University of Texas  Protein Translocation ecretory Pathway - Misfolded proteins in the ER N-linked glycosylation and ER protein folding - Misfolded proteins recognised by calnexin Molecular Biology of  Protein Translocation ecretory Pathway - Misfolded proteins in the ER Misfolded proteins Targeted for Degradation - Exported to the cytosol - Glycosylations removed - Poly-ubiquitinated - Degraded at the proteosome Molecular Biology of  Protein Translocation ecretory Pathway - Misfolded proteins in the ER Excess Misfolded proteins - Stimulates ER membrane proteins: S/T Kinase Inositol-Requiring enzyme 1 (IRE1) Protein kinase R-like ER kinase (PERK) Activating transcription factor 6 (ATF6) → Stimulate the Unfolded Protein response (UPR) Unfolded Protein response (UPR) - Reduce translation - Promotes expression of chaperone genes - Can lead to Apoptosis - regulated cell death Biology of Molecular  Protein Translocation sfolded Membrane Proteins - Pathogenicity Misfolded Membrane proteins Several fates: Mistargeted Degraded Upregulated - Lack of negative feedback Downregulated - Lack of positive feedback Unstable - Recycled internally Dysfunctional Cause disease Example 1: Cystic fibrosis, CFTR mutations Class III - Mistargeted/degraded Class IV - Dysfunctional Class VI - Unstable  Protein Translocation sfolded Membrane Proteins - Pathogenicity Misfolded Membrane proteins Several fates: Mistargeted Degraded Upregulated - Lack of negative feedback Downregulated - Lack of positive feedback Unstable - Recycled internally Dysfunctional Normal vs INSR Cause disease Example 2: Insulin-resistant syndrome, INSR defects - Reduced child weight gain and growth Kushi et al (2021) Diabetology  Protein Translocation sfolded Membrane Proteins - Pathogenicity Misfolded Membrane proteins Several fates: Mistargeted Degraded Upregulated - Lack of negative feedback Downregulated - Lack of positive feedback Unstable - Recycled internally Dysfunctional Cause disease Example 3: Autosomal Dominant Deafness type Reduced KCNQ4 2A, KCNQ4 mutations Increase function activity Decrease function/Less at the membrane Increase cellular K+ Hearing loss Protein Translocation and the Secretory Pathway Summary Protein Targeting Misfolded Membrane Proteins Mistargeted Eukaryotic Secretory Pathway Degraded Upregulated - Lack of negative Rough/Smooth ER recap feedback Downregulated - Lack of positive Protein Translocation feedback Co- and Post- translational Unstable - Recycled internally Dysfunctional Secretory Pathway - Modifications - Pathogenicity - Quality control Example 1: Cystic fibrosis, CFTR Chaperones, Calnexin defects - Misfolded Proteins Example 2: Insulin-resistant Poly-ubiquitinated and degraded at the syndrome, INSR defects proteosome Example 3: Autosomal Dominant IRE1, PERK and ATF6 Deafness type 2A, KCNQ4 mutations Protein Translocation and the Secretory Pathway Recommended Reading Molecular Biology of the Cell 6th Edition Chapter 12, p669-679; 685- Questions? Break  The Secretory Pathway Lecture Structure The secretory Exocytosis Pathway - Types of Secretion - Overview - Vesicle Delivery (RECAP) - Coated Vesicles - Neuronal Synapse Vesicle Delivery ER/Golgi Trafficking - Toxins on Vesicle Fusion - Vesicle Formation - Pathology in Vesicle Fusion - Vesicle Delivery - ER Protein Retrieval Endocytosis - Types Golgi Trafficking - The Golgi - Cis Golgi - Trans Golgi  The Secretory Pathway The secretory Pathway - Overview Protein Endoplasmic Translocatio Reticulum n Golgi Secretor Late y Endosome Vesicles Lysosom e Early Endosome Cell Exterior Molecular Biology of  The Secretory Pathway The secretory Pathway - Coated Vesicles Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo Steps in COPII Vesicle Formation 1) Sar1-GDP binds to a Sar1-GEF at the ER 2) Sar1 binds GTP → Sar1 exposes amphiphilic helix 3) Sar1 inserts into ER membrane Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo COPII adaptor coat Steps in COPII Vesicle Formation proteins 1) Sar1-GDP binds to a Sar1-GEF at the ER 2) Sar1 binds GTP → Sar1 exposes amphiphilic helix 3) Sar1 inserts into ER membrane 4) Sar1 binds COPII adaptor coat proteins (Sec23/24) Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo Steps in COPII Vesicle Formation 1) Sar1-GDP binds to a Sar1-GEF at the ER 2) Sar1 binds GTP → Sar1 exposes amphiphilic helix 3) Sar1 inserts into ER membrane 4) Sar1 binds COPII adaptor coat proteins (Sec23/24) 5) COPII coat protein complex (Sec13/31) binds 6) Transmembrane proteins recruited → membrane deforms 7) Membrane Molecular pinches off Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo - Not all vesicles are spherical Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo - Not all vesicles are spherical - Unfolded proteins are retained in the ER - Leaving the ER can be a selective using Exit Signals or happen by default bulk flow Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - Vesicle Formation ER to Golgi Movement - From ER exit sites (lack ribosomes) - COPII coated vesicles carry cargo - Not all vesicles are spherical - Unfolded proteins are retained in the ER - Leaving the ER can be a selective using Exit Signals or happen by default bulk flow - COPII coat is shed from final vesicles - Vesicles can fuse → Tubular Clusters - Vesicles or Vesicular Tubular Clusters Molecular transported Biology of along  The Secretory Pathway ER/Golgi Trafficking - Vesicle Delivery Vesicle Delivery Definitions Rab Protein - Ras-associated binding Protein - G Protein (guanine nucleotide-binding protein) SNARE - SNAP Receptor v-SNARE on vesicle t-SNARE on target membrane SNAP - Soluble NSF Attachment Protein NSF - N-ethylmaleimide sensitive factor Molecular Biology of  Molecular Biology of The Secretory Pathway the Cell ER/Golgi Trafficking - Vesicle Delivery Note: Similar pathway for any vesicle fusion with a target membrane. GDP dissociation inhibitor (GDI)  Molecular Biology of The Secretory Pathway the Cell ER/Golgi Trafficking - Vesicle Delivery Resetting Rab Proteins Resetting SNAREs Guanine nucleotide exchange factors N-ethylmaleimide-sensitive fusion protein (GEFs) (NSF) Rab-GDP → Rab-GTP SNAP to tether NSF to the membrane (Not shown) ATP  The Secretory Pathway ER/Golgi Trafficking - Vesicle Delivery Rab Proteins - Guide vesicles - >60 known types Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - ER Protein Retrieval Golgi to ER Movement - Retrieval of ER Proteins by “retrograde” transport - COPI coated vesicles carry cargo - Membrane protein retrieval - C-terminal Dilysine (KKxx or KxKxx) - Soluble ER retrieval signal - C-terminal KDEL Steps in COPII Vesicle Formation 1) KDEL signal recognised by the KDEL receptor 2) COPI coat assembles on KDEL receptor Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - ER Protein Retrieval Golgi to ER Movement - Retrieval of ER Proteins by “retrograde” transport - COPI coated vesicles carry cargo - Membrane protein retrieval - Dilysine - Soluble ER retrieval signal - KDEL Steps in COPI Vesicle Formation 1) KDEL signal recognised by the KDEL receptor 2) COPI coat assembles on KDEL receptor 3) Coat disassembles from vesicle 4) Vesicle transports back to ER 5) ER resident proteins released at the ER Molecular Biology of  The Secretory Pathway ER/Golgi Trafficking - ER Protein Retrieval Golgi to ER ER membrane protein complex subunit 3 Movement MAGPELLLDSNIRLWVVLPIVIITFFVGMIRHYVSILLQSDKKLTQEQVSDSQVLIRSRV Dilysine - ER membrane protein LRENGKYIPKQSFLTRKYYFNNPEDGFFKKTKRKVVPPSPMTDPTMLTDMMKGNVTNVLP retrieval MILIGGWINMTFSGFVTTKVPFPLTLRFKPMLQQGIELLTLDASWVSSASWYFLNVFGLR SIYSLILGQDNAADQSRMMQEQMTGAAMAMPADTNKAFKTEWEALELTDHQWALDDVEEE LMAKDLHFEGMFKKELQTSIF KDEL - Soluble ER protein HUMAN Endoplasmic reticulum chaperone BiP MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNR retrieval ITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVV EKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQ RQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNG VFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALS SQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIV LVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVC PLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLG TFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIER MVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEE KIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYGSAGPPPTGEEDTAEKDEL  The Secretory Pathway Golgi Trafficking - The Golgi Cis Golgi Network (CGN) Cis cisterna Medial cisterna Trans cisterna Trans Golgi Network (TGN) Molecular Biology of  The Secretory Pathway Golgi Trafficking - The Golgi Models of Golgi Organisation/Transport Molecular Biology of  The Secretory Pathway Golgi Trafficking - Cis Golgi Cis Golgi Function - Sorting of proteins/lipids to: 1) The ER 2) The Golgi cisterna - Phosphorylation of oligosaccharides on lysosomal proteins Lysosmal targeting disorders  The Secretory Pathway Golgi Trafficking - Trans Golgi Trans Golgi Function - Sorting of proteins/lipids to: 1) The Golgi cisterna 2) Endosomes 3) Secretory vesicles 4) The cell surface 5) Lysosomes (mannose 6- phosphate tag) - Sulfation of tyrosines and carbohydrates Molecular Biology of  The Secretory Pathway Golgi Trafficking - Trans Golgi Clathrin Coated Vesicles Golgi → Late Endosome Cell surface → Early Endosome Early Endosome → Golgi Molecular Biology of  The Secretory Pathway Golgi Trafficking - Trans Golgi Clathrin Coated Vesicles Molecular Biology of  The Secretory Pathway Golgi Trafficking - Trans Golgi Clathrin Coated Vesicles Dynamin GTPase in vesicle pinching Molecular Biology of  The Secretory Pathway Golgi Trafficking - Trans Golgi Clathrin Coated Vesicles Molecular Biology of  The Secretory Pathway Exocytosis & Endocytosis Molecular Biology of  The Secretory Pathway Exocytosis - Types of Secretion A) Constitutive secretion Proteins with no specific targeting signal are secreted to the cell’s surface Examples: - Plasma membrane proteins - Soluble proteins B) Regulated secretion Vesicles can pack and retain specific cargo until signalled for release. Examples: - Neurotransmitter Molecular Biology of secretion  The Secretory Pathway Exocytosis - Vesicle Delivery RECAP Vesicle Delivery Definitions Rab Protein - Ras-associated binding Protein - G Protein (guanine nucleotide-binding protein) SNARE - SNAP Receptor v-SNARE on vesicle t-SNARE on target membrane SNAP - Soluble NSF Attachment Protein NSF - N-ethylmaleimide sensitive factor Molecular Biology of  The Secretory Pathway Neuronal Synapse - Vesicle Delivery Synapse Vesicle Delivery SNARE - SNAP Receptor v-SNARE on vesicle Synaptobrevin t-SNARE on target membrane Syntaxin SNAP - Soluble NSF Attachment Protein Schwarz Fundamental Neuroscience (4th Edition,  The Secretory Pathway Exocytosis - Toxins on Vesicle Fusion Synapse Vesicle Delivery SNARE - SNAP Receptor v-SNARE Synaptobrevin t-SNARE Syntaxin SNAP - Soluble NSF Attachment Protein Clostridial Neurotoxins (CNTs) Tetanus toxin - Cuts Synaptobrevin Botulinum neurotoxins (Seven types) - Cut SNARES and SNAP-25 Schwarz Fundamental Neuroscience (4th Edition,  The Secretory Pathway Exocytosis - Pathology Many diseases are associated with incorrect vesicle trafficking Yarwood et al (2020) Disease Models & Mechanisms  The Secretory Pathway Exocytosis - Pathology Vesicle fusion defects Example 1: Congenital muscular dystrophy (CMD) BET1 deficiency or mutation → Reduced COPII vesicle fusion → Reduced muscle function Symptoms Low muscle tone or floppiness (hypotonia) Respiratory problems Tightness of the ankles, hips, knees and elbows Eye problems Donkervoort et al (2021) EMBO Mol Med Learning difficulties 13:e13787  The Secretory Pathway Exocytosis - Pathology Vesicle fusion defects Example 2: Cranio-lenticulo-sutural dysplasia Autosomal recessive syndrome SEC23A mutation → abnormal ER-to-Golgi trafficking → late-closing fontanels → facial dysmorphisms Symptoms Skull hypomineralization Skeletal defects Y-shaped cataracts Hypertelorism - large distance between (eyes) Boyadjiev et al (2006) Nature Genetics 38:1192–1197  Endocytosis Endocytosis - Transport Pathways Molecular Biology of  Endocytosis Endocytosis - Types Clathrin coated pit, vesicle formation A) Pinocytosis - Endocytic vesicles constantly forming - Coupled with exocytosis to maintain cell size 1) Clathrin pit mediated - Specific cargo receptors Example: LDL Receptor Molecular Biology of  Endocytosis Endocytosis - Types A) Pinocytosis - Endocytic vesicles constantly forming - Coupled with exocytosis to maintain cell size 1) Clathrin pit mediated Clathrin-independent 2) Clathrin-independent pinocytosis - Take in extracellular fluid/material i) Actin/Dyamin motion ii) Caveolin iii) Clathrin-independent carrier (CLIC) Molecular Biology of  Endocytosis Endocytosis - Types A) Pinocytosis - Endocytic vesicles constantly forming - Coupled with exocytosis to maintain cell size 1) Clathrin pit mediated 2) Clathrin-independent 3) Macropinocytosis - Induced for a limited time - Cell-surface protrusions "ruffles" - Large fluid-filled endocytic vesicles formed Molecular Biology of  Endocytosis Endocytosis - Types B) Phagocytosis Bacterium Bacterium - Surface receptors detect cargo - Pseudopod extension with actin - Engulf large particles (e.g. Bacterial/Virus) → Form a “phagosome” PI(4,5)P2 - Stimulates actin polymerization → Pseudopod extensions form PI(3,4,5)P3 - Depolymerizes actin filaments at the base In the cell: - Phagosomes fuse with lysosomes Molecular Biology of  The Secretory Pathway Summary The secretory Pathway Exocytosis - Overview - Types of Secretion - Constitutive or Regulated - Coated Vesicles - Vesicle Delivery (RECAP) - Neuronal Synapse Vesicle Delivery ER/Golgi Trafficking v-SNARE - Synaptobrevin - Vesicle Formation - COPII Vesicles t-SNARE - Syntaxin Sar1, Adaptor coat proteins - Toxins on Vesicle Fusion - Tetanus & Botulinum (Sec23/24) - Pathology in Vesicle Fusion - CMD & Cranio- Coat protein complex lenticulo-sutural dysplasia (Sec13/31) Transmembrane proteins Endocytosis - Vesicle Delivery - Rab/v-SNARE/t- A) Pinocytosis SNARE 1) Clathrin pit mediated - ER Protein Retrieval - COPI 2) Clathrin-independent - Actin/Dyamin, Vesicles Caveolin, CLIC - Dilysine/KDEL motifs 3) Macropinocytosis B) Phagocytosis  The Secretory Pathway Recommended reading Molecular Biology of the Cell 6th Edition ER To Golgi - Chapter 13, p710-716, p720-722 Exocytosis - Chapter 13, p741-750 Endocytosis further reading: Rennick Endocytosis et al (2021)- Nature Chapter 13, p729-740 Nanotechnology 16:266–276 Questions?