Intracellular Transport Lecture Notes (HC_luirink_2024) PDF

 Lecture (Intracellular Transport) Aims obtain insight how proteins and lipids are transported in the cell to reach their destination -appreciate general principles in “protein and lipid trafficking” -mechanistic insight in biogenesis of membrane proteins -...

 Lecture (Intracellular Transport) Aims obtain insight how proteins and lipids are transported in the cell to reach their destination -appreciate general principles in “protein and lipid trafficking” -mechanistic insight in biogenesis of membrane proteins -understand relationship between problems in “protein trafficking” and disease  Course Aims At the end of this course you will be able to: -explain the relation between the shape and the function of cells. -explain how cells together form a tissue, with the digestive system as an example. -analyse histological preparations using light microscopy. -draw the structure and organelles of the cell, and explain their role in functioning of the cell. -analyse ultrastructures of the cell using data derived form the electron microscope. -explain how intracellular proteins and lipids are transported to reach their subcellular destination. -describe the different signal transduction pathways and layers of organization that underlie the regulation of cellular processes. -mathematically model the connectivity of signal transduction pathways.  Lectures/Workgroups Intracellular Transport and Exam Material Lectures Book: Essential Cell Biology, 4th or 5th or 6th ed. (Alberts) Material: Chapter 15 complete; chapter 11, pages 369-374 (4th ed.) or 375-380 (5th ed.) or 390-396 (6th ed.) plus figs from other books (see canvas: HC_luirink_2024) Workgroups Prepare: -questions (canvas: Questions_workgroup_Luirink.2024.doc) Read: -comment Nobelprize 1999, 2013, 2017, 2024 (canvas) Watch: -videos Questions: -about the content presented in the lectures  How are functions in the cell separated? large enzyme complexes (prokaryotes and eukaryotes) compartmentalization of enzymes in organelles (eukaryotes) surrounded by membrane: How do proteins/lipids get to the organelles (signals/receptors)? How do proteins/lipids move between organelles? How do proteins cross membrane of organelle? How do proteins fold in organelle? How do proteins insert in a membrane, fold and function in the membrane? How is the quality of these processes controlled? Intracellular transport in pro- and eukaryotes Cell organelles (EM) In nucleus: RNA/DNA synthesis In cytoplasm: -cytosol (protein synthesis/degradation -organelles (specific functions)  Cell organelles (scheme) Organel has lumen and membrane Specific lipid and protein in lumen and membrane (need for sorting) Organel has same function in all cells Organel has specific position in cell Abundance of organel related to cell type  Evolution of ER/GA/endosomes/lysosomes/nucleus/peroxisomes) Pinching off from plasma membrane  Evolution of mitochondria/chloroplasts Endosymbiosis (uptake of bacteria); necessary for larger cells (higher surface/volume ratio) Transport mechanisms Large pores: cytosol nucleus Narrow channels: cytosol --> ER/mitoch./chloropl./ perox. Vesicles: ER GA Endosome/lysozome/ PM Roadmap of protein traffic Large pores Narrow pores No pores, vesicles  Signal sequences 3-30 aminoacids long Also called targeting or sorting signals used to target proteins to a specific organel Recognised by receptor -->targeting to destination Necessary and sufficient for sorting! Signal sequence switching --> necessary and sufficient for sorting Roadmap to nucleus Nuclear envelope Relatively large  Nuclear Pore Function: -nuclear export: RNA, ribosomal subunits -nuclear import: nuclear proteins with NLS signal NB Transport is active (requires GTP) and the transported proteins are folded! Signal sequences Import in nucleus Import in nucleus Roadmap to mitochondria and chloroplasts  Transport into mitochondria ATP synthesis derived from bacteria; still contain DNA/ribosomes part of proteins synthesized in organel, part imported from cytosol subcompartments  Import in mitochondria synth. in cytosol with mit. ss binding to cytosolic chaperones/targeting factors ss binds to receptor in OM Translocation at contact sites binding to matrix chaperones (pulling/folding) cleavage of ss further sorting NB Post-translational process! Signal sequences Subcompartments in chloroplasts lumen is called stroma extra compartment: tylakoid -requires extra ss -different mechanisms of targeting Roadmap to peroxisomes peroxisomes oxidation of fatty acids single membrane no DNA/ribosomes Peroxisomal import synthesis with PTS binding to receptor in cytosol binding to receptor in membrane translocation through membrane channel cytosolic receptor returns to cytosol NB transport in folded form! Signal sequences Roadmap to ER  Endoplasmic Reticulum (ER) network ER is continuous network  Two types of ER RER (rough endoplasmic reticulum): central place of protein and lipid synthesis; rough appearance due to ribosomes SER (smooth endoplasmic reticulum): specialised functions (detoxification, production of steroid hormones, storage of Ca) Two sites of protein synthesis Free ribosomes: -cytosolic proteins -mit./chlor./perox. proteins post-translational process Membrane-bound ribosomes: all proteins of secretory pathway (ER/GA/lysosomes/ endosomes/PM) co-translational process  Co- versus post-translational translocation all proteins of secretory mit./chlor./perox. pathway(ER/GA/lysosomes/ Proteins; no ER signal endosomes/PM); starts at ER sequence! With ER signal sequence! Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)  Signal sequences ER signal sequence first discovered (in vitro system by Blobel) N-terminal; +/- 20 aa with hydrophobic core Directs transport to ER, is cleaved in ER Functions: -targeting to ER via binding of SRP! -opening of translocation channel  The Signal Recognition Particle (SRP) SRP is ribonucleoprotein complex SRP consists of 6 proteins and RNA SRP binds ss of nascent proteins, causes translation arrest and binds to SRP receptor  Details of co-translational translocation Synthesis with ss SRP binds SRP receptor Binding of SRP SRP dissociates from ss Pause in translation (translation starts Targeting to ER again) membrane Co-translational translocation Figure 12-40 Molecular Biology of the Cell (© Garland Science 2008)  Co-translational translocation of soluble proteins End-stage of translocation: ss cleaved and degraded; mature protein folds and can be further sorted Figure 12-38 Molecular Biology of the Cell (© Garland Science 2008)  Membrane proteins, key questions ✓ How do they get there (targeting)? ✓ How do they insert into the membrane? ✓ How do they fold, assemble and degrade?  Why membrane proteins ? ✓ 30% of sequenced genes encode membrane proteins ✓ membrane proteins are involved in vital cellular processes (signal transduction, communication, transport) ✓ 70% of drug targets are membrane proteins ✓ many diseases due to compromised folding of membrane proteins (Cystic Fibrosis, Alzheimer) ✓ very few high resolution structures available  Membrane(-associated) proteins Transmembrane proteins: single-, double- or multi-pass Trans membrane helix Transmembrane proteins are anchored in lipids via hydrophobic -helices  Insertion of single-pass membrane protein (2 signals) One signal for targeting, one signal for anchoring End topology: N-lumen/C-cytosol  Insertion of single-pass membrane protein (1 signal) One signal for targeting and anchoring Topology determined by positive-inside rule (inside means cytosolic) Insertion of double-pass membrane protein Topology of multi-pass membrane proteins ++ ++ ++ Fate of proteins that have entered the ER (soluble and membrane proteins!) Stay in ER via ER retention signals (see later) Further routing in secretory pathway via vesicular transport Roadmap of vesicular traffic  Two directions in vesicular traffic 1. Endocytic pathway: uptake of material from exterior - -> degradation in lysosomes --> metabolites to cytosol 2. Secretory pathway: flow of material towards exterior (modification/storage/exocytosis of proteins)  Exocytosis and endocytosis Figure 13-1 Molecular Biology of the Cell (© Garland Science 2008)  Characteristics of vesicular traffic Transport of both soluble and membrane proteins (“cargo”) Different types of vesicles to ensure correct sorting (right cargo to right organel) Transport must be specific and well-regulated  Vesicle budding Vesicle has protein coat Function of coat: -1. shape to facilitate budding (triskelions) -2. capture molecules to transport  1. Coat gives shape (round basket) Clathrin (example of coat) consists of triskelions --> football-like structures that pinch off from donor membrane  2. Coat selects cargo Different adaptins dependent on donor membrane Specificity of transport (where do the naked vesicles go) Vesicles move between compartments via motor proteins along cytoskeleton Initial recognition by Rab proteins that are recognized by tethering proteins Further specificity by v-SNARE in vesicle that docks at t-SNARE in target membrane; many different SNAREs for specific fusion Specificity of transport (where do the naked vesicles go) SNARES induce membrane fusion  Secretory pathway Secretory pathway starts at ER --> GA --> PM --> external milieu All transport in secretory pathway via vesicles During transport: 1. modification (folding, glycosylation) 2. quality control (retention and degradation of misfolded proteins) 3. sorting NB This starts already in ER  1. Modification f.i. glycosylation in lumen of ER Precursor oligosaccharide transferred to Asn in Asn-X-Ser/Thr motif Glycosylation during synthesis Trimming and processing of sugars continues in ER and GA --> structure, protection, folding and sorting  Secretory pathway Secretory pathway starts at ER --> GA --> PM --> external milieu All transport in secretory pathway via vesicles During transport: 1. modification (folding, glycosylation) 2. quality control (retention and degradation of misfolded proteins) 3. sorting NB This starts already in ER  2. Quality control in the ER Only correctly folded proteins can leave the ER Unfolded proteins remain complexed with chaperones --> folding or transfer to cytosol for degradation NB This is sometimes detrimental (f.i. cystic fibrosis) Unfolded protein response Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  Secretory pathway Secretory pathway starts at ER --> GA --> PM --> external milieu All transport in secretory pathway via vesicles During transport: 1. modification (folding, glycosylation) 2. quality control (retention and degradation of misfolded proteins) 3. sorting NB This starts already in ER  3. Sorting Proteins that have entered the ER can either stay there or move on towards the GA Proteins “move on” by default; they need special mechanisms to stay in the ER A. Aggregation or interaction with chaperones (prevents packaging in vesicles) B. Retention in ER via recognition of retention signal in the protein (KDEL or KKXX)  ER retention sequence is a targeting sequence B. Retention in ER via recognition of retention signal in the protein (KDEL or KKXX) Retrieval of ER resident proteins, mechanism, part I Retrieval of ER resident proteins, mechanism, part II All ER resident proteins have the retention signal; if they “escape” to the GA, they are recognised by a receptor and shuttled back to the ER Roadmap from ER to GA  The Golgi Apparatus, structure Stacks of cisternae and tubules GA is polarised (cis-side faces the ER, trans-side faces the PM  The Golgi Apparatus, function Modification of proteins, primarily trimming of sugars (glycosylation) Functional compartmentalization of the GA CGN and TGN are sorting platforms Cisternae form the “powerplant” of glycosylation NB different enzymes in different subcompartments; trafficking between subcompartments via vesicles Roadmap of exocytosis Two pathways of exocytosis  Two pathways of exocytosis: characteristics Constitutive secretion: -default, continuous process -present in all cells -permanent flow of material to membrane (f.i. extracellular matrix proteins Regulated secretion: -dependent on stimulus (f.i. hormone) -present in specialised cells -storage in vesicles near PM NB During exocytosis PM increases in volume but this is compensated by endocytosis (see later) Secretory vesicles store and release concentrated proteins Roadmap of endocytosis Endocytosis and degradation A. Phagocytosis: uptake of large particles B. Pinocytosis: uptake of solutes 1. ordinary pinocytosis 2. receptor-mediated endocytosis (RME)  Endocytosis: A. phagocytosis Phagocytosis: uptake of large particles Uptake of bacteria (defense mechanism) or cellular debris; by specialised cells (macrophages) Particles usually bind to receptors (f.i. for antibodies) before uptake (”triggered process”) Uptake --> large endocytic vesicles (phagosomes) --> fusion with lysosomes --> degradation Example of phagocytosis: uptake of bacterium Example of phagocytosis: uptake of red blood cell  Endocytosis: B. pinocytosis Pinocytosis: uptake of solutes (small stuff) Uptake via clathrin coated pit-->clathrin coated vesicle --> uncoating--> fusion with early endosome --> late endosome--> lysosome  Endocytosis: B1. ordinary pinocytosis Pinocytosis: uptake of solutes (small stuff) Indiscriminate continuous uptake --> retrieval of membrane material  Endocytosis: B2. Receptor-mediated endocytosis (RME) Pinocytosis: uptake of solutes (small stuff) Specific binding of macromolecules to receptors --> accumulation in clathrin coated pits --> uptake  Example of RME: LDL Cholesterol in blood in LDL-->binds LDL receptor -->concentration in coated pit --> uptake -->endosome --> dissociation of LDL- receptor complex by low pH --> transfer to lysosome --> LDL is degraded/receptor returns to PM NB These systems are often hijacked by viruses (f.i. HIV) RME: hijacked by viruses  Sorting in endosomes Early endosomes --> fusions to form larger (late) endosomes --> develop into or fuse with lysosomes Gradual decrease in pH (lowest in lysosomes) Receptors are often released at low pH --> recycling, degradation or transcytosis Cargo always goes to lysosome for degradation Roadmap to lysosomes Lysosomes Degradation of macromolecules by acid hydrolases Hydrolases have low pH optimum One membrane: -protects cytosol -contains many transporters -contains ATPase NB Membrane proteins are highly glycosylated for protection  4 entry pathways for lysosomes 2 1. Pinocytosis/endocytosis 1 2. Phagocytosis 3. Autophagy 3 4. Vesicular traffic from GA 4  4. Vesicular traffic from GA to lysosomes Patch ss in hydrolase is recognised and M6P is attached in CGN M6P binds to M6P receptor in TGN Packaging in clathrin coated vesicles Delivery of cargo (hydrolase) at lysosome Dissociation of M6P from receptor by low pH --> recycling of receptor Removal of phosphate from hydrolase --> active mature enzyme Roadmap of protein traffic Large pores Narrow pores No pores, vesicles