17/01/2017. Protein trafficking between cell compartments. Lecture 3: The cytosol. The mitochondrion - the power plant of the cell

Transcription

1 ell biology 2017 version 13/ ote endosome vs lysosome handout Lecture 3: Text book Alberts et al.: hapter (Topics covered by the lecture) A lot of reading! Focus on principles ell Biology interactive media video or interactive 1 Protein trafficking between cell compartments ytosol ucleus Ribosomes Mitochondrion >90 % of all membranes are part of organelles within the cytoplasm Ribosome populations Free cytosolic Attached to the endoplasmic reticulum Protein Various address tags (without a tag cytosol) 2 The cytosol Viscous solution high concentration of proteins (~400 mg/ml) Glucose ucleus ATP (2) ADH (2) Pyruvat 3 The mitochondrion - the power plant of the cell From Greek, mitos, thread, chondros, granule The mitochondrion is a double membrane-enclosed organelle that specialize in ATP regeneration (>100 per cell) 1 m 0.5 m The invaginations are denoted cristae increased surface area Key processes/ components of the cytosol Translation Signal transduction Glycolysis Reproduce by dividing in two (binary fission) 4 Metabolic pathways of the mitochondrion Intermediary metabolism Pyruvate Acetyl oa Anim. 05-citric_acid_cycle.mov (5 min) Fatty acid Acetyl oa Krebs cycle Respiration (electron transport chain) and ATP synthesis ADH The origin of the mitochondrion and its genome Aerobic bacteria Founding eukaryote Mitochondria have circular DA and bacteria-like ribosomes 37 genes 22 tra genes 2 rra genes 13 mra encoding genes Anim. 13-electron_transport & 14-ATP_synthase H gradient and utilization of its energy for ATP production. 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

2 Targeting proteins to the mitochondrion Protein translocation across the mitochondrial membranes is mediated by proteins that form a channel spanning both membranes Endoplasmic reticulum network within cytoplasm Protein sorting and modification (Rough ) (starting point of the secretory pathway of protein synthesis) Lipid synthesis (Smooth ) Detoxifications (Smooth, eg. P450) Translocation of mitochondrial proteins through this channel requires proteins to be kept unfolded Folded protein Unfolded protein a 2 storage (Smooth ) o passage Anim. 13-protein_import Successful passage Signal sequence haperone, keeping the protein unfolded in cytosol 7 Lipid = Lipid 8 Protein targeting to the endoplasmic reticulum o-translational protein translocation Anim translation-I ytosolic ribosome Ribosome Signal-recognition particle (SRP) associated ribosome SRP receptor mra tra signal sequence, stretch of hydrophobic a.a. lumen Protein translocator Protein is translocated into the lumen of the co-translationally Signal sequence is cleaved by a peptidase after completion of translation/ translocation Pause in translation during localization step 9 10 Integration of a transmembrane protein into 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 membrane ote the opening of the protein translocater, which allows lateral diffusion within the membrane of both the -signal sequence and trans-membrane domain

3 Synthesis of multi-pass transmembrane proteins Re-start-transfer sequence - Stop-transfer sequence signal sequence (-terminus)= SRP the initial start transfer signal followed by a signal peptidase recognition site entry: the first step to various destinations 14 Post office Out of the cell (secretion) Plasma membrane Secretory pathway January 2017 Translocation stop and re-start several times, which results in a multi-pass transmembrane protein 13 Anim. 16-protein_translocation.mov Proteins are glycosylated during passage of the secretory pathway Glycocalyx a carbohydrate zone on the cell surface Extracellular Post-translational modification by attachment of oligo-saccharides ytosol -linked oligo-saccharides are attached via the amide group of asparagine in H O-linked oligo-saccharides are attached to hydroxyl group of serine or threonine in Vesicular trafficking post Post office Out of the cell Plasma membrane Secretory pathway Transport from to, within, and from to either lysosomes or cell surface is carried out by transport vesicles (liposomes made of phospholipids) O 15 Video 12-biosy_secret_path 16 The architecture of the apparatus Proteins that keep the cisterna together ucleus Principle of vesicular transport Donor compartment 3-10 cisterna (containing different sets of processing enzymes) Trans-face is-face Budding of vesicle from donor compartment Vesicle transport The cytoskeleton is used often used as railway tracks Downstream target compartments Docking and fusion of a vesicle with its target compartment Transport vesicles 17 Target compartment 18 3

4 17/01/2017 Vesicle formation in donor compartment Different coating proteins in vesicular trafficking Vesicle pinching off Endocytosis at the plasma membrane oat: Vesicle formation lathrin OPI oat Bud formation protein Sorting receptor argo (i.e., the protein to be transported) OPII argo onstricting protein complex Adaptin Sorting receptor Lumen of donor compartment Vesicle docking and fusion with target compartment Tethering of vesicles to the correct target compartment Uncoating of vesicle subsequent to pinching off Rab protein on vesicle docks with Rab effector on target compartment Rab protein Vesicle tethering with target compartment (specificity Rab s) Vesicle docking and fusion with target compartment (SAREs) Rab effector (tethering protein) Different Rab proteins different target compartments ompartment X ompartment Y Lumen of target compartment Fusion of a vesicle with its target compartment Protein trafficking in the vesicular pathway v-sare Plasma membrane lathrin t-sare SARE proteins on vesicle and target compartment interacts OPI Retrieval of proteins (KDEL receptor) Anterograde transport onformational changes of SAREs bring the membranes closer together.. OPII Retrograde transport..until they are in physical contact This leads to exclusion of H2O membrane fusion 23 Endoplasmic reticulum 24 4

5 onstitutive Protein trafficking: post- Exocytosis Regulated (e.g. insulin) Lysosomal pathway A Primary lysosome B Endosome Secondary lysosome The term lysosome defines a function: lys: digest soma: body The lysosome the digestive system of the cell Vesicles (~ 300/cell) filled with ~ 40 acid hydrolases that has capacity to degrade more or less anything The lysosome is responsible for degradation of exogenous and endogenous macromolecules and structures The inside of the lysosome is acidic ph 7.2 Anim. 11- clathrin Video 14- endosome_ fusion A B s develop from endosomes by fusion with vesicles carrying lysosomal enzymes m ph 5 H H H ATP ADP H P 26 The ph regulates the activity of hydrolytic enzymes contains many types of hydrolytic enzymes These are only active in an acidic environment Uptake of material from the exterior Phagocytosis ( cell eating ) specific uptake of large (0.5 2 m) particles, primary by immune cells Receptor-mediated endocytosis - specific uptake of molecules on -specific endocytosis, pinocytosis ( cell drinking ) - anything small in the extracellular fluid is taken up indiscriminately Hydrolases are inactive in and (ph ~7) Hydrolases are active in the acidic lumen of the lysosome Degradation of endocytosed material 28 Hydrolases: proteases, nucleases, phosphatases etc etc. 27 video 15 phagocytosis.mov Three routes to the lysosome Phagocytosis Endocytosis 5. Autophagy Phagosome Endosome Autophagosome Primary lysosome 5. Secondary lysosome 29 Anim. 13-receptor_endocytosis (ote: vesicle fusion with endosome) Summary: cellular organelles and trafficking 3 types of protein transport A. Gated (nuclear pores) B. Across membranes** (translocation channels). Vesicle (budding and fusion) ytosol (54%) >10-fold more internal membranes than plasma membrane % = volume of a liver cell ** ucleus (6%) ** Ribosomes (1%) (12%) (3%) Mitochondrion (22%) Endosome (1%) 30 5