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Download Lecture 3: Protein trafficking between cell compartments The cytosol
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17/01/2017 Cell biology 2017 Protein trafficking between cell compartments version 13/1 2017 Note – endosome vs lysosome handout Lecture 3: Free cytosolic Ribosome populations Nucleus ER Golgi Text book Alberts et al.: Chapter 12 - 14 (Topics covered by the lecture) Protein N Ribosomes Cytosol Attached to the endoplasmic reticulum C Lysosome A lot of reading! Focus on principles Mitochondrion >90 % of all membranes are part of organelles within the cytoplasm 1 Cell Biology interactive media ”video” or ”interactive” 3 The cytosol Viscous solution high concentration of proteins (~400 mg/ml) The mitochondrion - the power plant of the cell • From Greek, mitos, thread, + chondros, granule 1 m ATP (2) Nucleus 2 • The mitochondrion is a double membrane-enclosed organelle that specialize in ATP regeneration (>100 per cell) Glucose 2. 1. Various address tags (without a tag cytosol) NADH (2) 0.5 m Pyruvat The invaginations are denoted cristae increased surface area 3. Key processes/ components of the cytosol 1. Translation 2. Glycolysis + 3. Signal transduction 4 Metabolic pathways of the mitochondrion 1. Intermediary metabolism • Reproduce by dividing in two (binary fission) Pyruvate Fatty acid Acetyl CoA Acetyl CoA Anim. 02.5-citric_acid_cycle.mov (1.5 min) Krebs cycle NADH 2. Respiration (electron transport chain) and ATP synthesis The origin of the mitochondrion and its genome + Aerobic bacteria ”Founding” eukaryote • Mitochondria have circular DNA and bacteria-like ribosomes 22 tRNA genes 1. 37 genes 2. 2 rRNA genes 13 mRNA encoding genes H+ gradient and utilization of its energy for ATP production. Anim. 14.3-electron_transport & 14.4-ATP_synthase • Mitochondria are only inherited from the mother • Most of the mitochondrial proteins are encoded in the nucleus and have to be imported from the cytosol 6 1 17/01/2017 Endoplasmic reticulum – ”network within cytoplasm” Targeting proteins to the mitochondrion 1. Protein sorting and modification (Rough ER ) (starting point of the “secretory pathway” of protein synthesis) 2. Lipid synthesis (Smooth ER) Protein translocation across the mitochondrial membranes is mediated by proteins that form a channel spanning both membranes 3. Detoxifications (Smooth ER, eg. P450) 4. Ca2+ storage (Smooth ER) Translocation of mitochondrial proteins through this channel requires proteins to be kept unfolded Folded protein 1. Unfolded protein N Signal sequence No passage Successful passage Anim. 12.3-protein_import Chaperone, keeping the protein unfolded in cytosol 7 Protein targeting to the endoplasmic reticulum Anim. 06.6-translation-I 3. 2. L+i+p+i+d = Lipid 4. 8 Co-translational protein translocation ER associated ribosome SRP receptor Cytosolic ribosome Signal-recognition particle (SRP) Ribosome mRNA tRNA ER lumen ER signal sequence, stretch of hydrophobic a.a. Protein is translocated into the lumen of the ER co-translationally Protein translocator Signal sequence is cleaved by a peptidase after completion of translation/ translocation 9 Pause in translation during localization step 10 Integration of a transmembrane protein into ER C N Translocation is initiated but stops at a hydrophobic ~15 aa sequence termed stoptransfer signal However, translation continues Translation complete the stop-transfer signal sequence integrates into the ER membrane Note the opening of the protein translocater, which allows lateral diffusion within the ERmembrane of both the ER-signal sequence and trans-membrane domain 12 11 2 17/01/2017 Synthesis of multi-pass transmembrane proteins Re-start-transfer sequence ER entry: the first step to various destinations 14 ER signal sequence (N-terminus)= the initial ”start transfer signal” followed by a signal peptidase recognition site CStop-transfer sequence Post office Plasma membrane ER ER SRP Out of the cell (secretion) Lysosome Golgi Secretory pathway January 2017 Translocation stop and re-start several times, which results in a multi-pass transmembrane protein 13 Anim. 12.6-protein_translocation.mov Proteins are glycosylated during passage of the secretory pathway Vesicular trafficking post ER “Glycocalyx – a carbohydrate zone on the cell surface” Extracellular Post-translational modification by attachment of oligo-saccharides O-linked oligo-saccharides are attached to hydroxyl group of serine or threonine in Golgi O Golgi Lysosome Secretory pathway Transport from ER to Golgi, within Golgi, and from Golgi to either lysosomes or cell surface is carried out by transport vesicles (liposomes made of phospholipids) Video 13.2-biosy_secret_path Principle of vesicular transport Nucleus Donor compartment 1. Budding of vesicle from donor compartment 3-10 Golgi cisterna (containing different sets of processing enzymes) Downstream target compartments Golgi 16 15 The architecture of the Golgi apparatus Trans-face Out of the cell Plasma membrane ER Cytosol N-linked oligo-saccharides are attached via the amide group of asparagine in ER H N ER Proteins that keep the Golgi cisterna together Post office The cytoskeleton is used often used as railway tracks 2. Vesicle transport Cis-face 3. Docking and fusion of a vesicle with its target compartment ER Transport vesicles Target compartment 18 17 3 17/01/2017 Vesicle formation in donor compartment Different coating proteins in vesicular trafficking 3. Vesicle pinching off Endocytosis at the plasma membrane Coat: 2. Vesicle formation Clathrin COPI Coat 1. Bud formation protein Sorting receptor Cargo (i.e., the protein to be transported) COPII Cargo Constricting protein complex Lysosome Adaptin Sorting receptor Golgi Lumen of donor compartment ER 19 20 Vesicle docking and fusion with target compartment Tethering of vesicles to the correct target compartment 1. Uncoating of vesicle subsequent to ”pinching off” Rab protein on vesicle docks with Rab effector on target compartment Rab protein 2. Vesicle tethering with target compartment (specificity Rab’s) 3. Vesicle docking and fusion with target compartment (SNAREs) Rab effector (tethering protein) Different Rab proteins – different target compartments 1. Compartment X 2. 3. Compartment Y Lumen of target compartment Fusion of a vesicle with its target compartment Protein trafficking in the vesicular pathway v-SNARE 1. 22 21 Plasma membrane 2. Lysosome 3. Clathrin Golgi t-SNARE 1. SNARE proteins on vesicle and target compartment interacts COPI Retrieval of ER proteins 4. (KDEL receptor) Anterograde transport 2. Conformational changes of SNAREs bring the membranes closer together….. COPII Retrograde transport 3. …..until they are in physical contact 4. This leads to exclusion of H2O membrane fusion 23 Endoplasmic reticulum 24 4 17/01/2017 Protein trafficking: post-Golgi Exocytosis Regulated Constitutive (e.g. insulin) The lysosome – the digestive system of the cell Lysosomal pathway A Primary lysosome • Vesicles (~ 300/cell) filled with ~ 40 acid hydrolases that has capacity to degrade more or less anything B Endosome • The lysosome is responsible for degradation of exogenous and endogenous macromolecules and structures C Secondary lysosome The term lysosome defines a function: lys: digest soma: body pH 7.2 Lysosomes develop from endosomes by fusion with vesicles carrying lysosomal enzymes B A Anim. 13.1clathrin • The inside of the lysosome is acidic 0.2-0.5 m H+ pH 5 H+ H+ C Video 13.4endosome_ fusion ATP 25 H+ The pH regulates the activity of hydrolytic enzymes Lysosome contains many types of hydrolytic enzymes These are only active in an acidic environment + ADP + P 26 Uptake of material from the exterior 1. Phagocytosis (“cell eating”) – specific uptake of large (0.5 – 2 m) particles, primary by immune cells 2. Receptor-mediated endocytosis - specific uptake of molecules + 3. Non -specific endocytosis, pinocytosis (“cell drinking”) - anything small in the extracellular fluid is taken up indiscriminately Degradation of endocytosed material Hydrolases are inactive in ER and Golgi (pH ~7) 28 1. Hydrolases are active in the acidic lumen of the lysosome 27 Hydrolases: proteases, nucleases, phosphatases etc etc. video 13.5 phagocytosis .mov Three routes to the lysosome Summary: cellular organelles and trafficking 3 types of protein transport A. Gated (nuclear pores) B. Across membranes** (translocation channels) C. Vesicle (budding and fusion) Cytosol (54%) Phagocytosis 1. Endocytosis 4. 2. 4. 5. 4. ER 3. Autophagy 1. Phagosome 2. Endosome 4. Primary lysosome Anim. 13.3-receptor_endocytosis 3. 2. >10-fold more internal membranes than plasma membrane 3. Autophagosome 5. Secondary lysosome (Note: vesicle fusion with endosome) 29 % = volume of a liver cell Nucleus (6%) ER (12%) Golgi (3%) ** Ribosomes ** Lysosome (1%) Mitochondrion (22%) Endosome (1%) 30 5