Transcript Document
Vesicle formation and targeting is a multi-step process 2. Formation of coated transport vesicle… 3. Targeting and docking to specific compartment… SNAREs and Rabs Target compartment 1. Formation of coated buds… (ATP, GTP, and cytoplasmic protein factors…) GTP GDP + Pi 4. Uncoating… GAP and Sar1 GEF and Sar1 Donor compartment Coat proteins (“COPs” or “coatomer”) 5. Fusion… SNARE plus other fusion proteins Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting/targeting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome How are proteins sorted to appropriate vesicles so that they are transported to proper location? What are the address labels? Two secretory pathways; constitutive and regulated Default pathway for ER/Golgi proteins If no address label, then secrete ECB 15-28 Signal required to trigger secretory granule fusion Inside lumen is equivalent Example - neurotransmitter to outside of cell release Regulated secretion Secretory granules containing insulin in pancreatic cells Signal for release is elevated glucose levels in blood If secretion is default, how are resident ER proteins retained? Ex: BiP is a member of the HSP70 family that functions in the ER BiP They aren’t! KDEL Constituitive secretion KKXX KDEL-R Secretory granule Regulated secretion ER CGN C, M, T Golgi TGN Plasma membrane Outside BiP escapes from ER and must be “retrieved” from the Golgi… C-terminal KDEL in BiP sequence functions as retrieval signal… KDEL-receptors in Golgi direct retrieval/recycling… KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER… Summary so far of protein targeting, revisited… Protein targeting Secretion/membrane proteins Cytoplasm Signal sequence (hydrophobic a-helix) Vesicle targeting RER See ECB figure 14-5 Default KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default ? (constituitive secretion) Plasma membrane Transport Lysosomes Retrieval How are proteins targeted to the lysosome? Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesicles SNAREs Protein sorting Secretion - Golgi to plasma membrane Retention in ER Golgi to lysosome How are proteins sorted to vesicles leaving TGN for lysosome? Lysosomes degrade and recycle macromolecules… ECB 15-34 Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromolecules pH of lumen is about 5 - acidic! How are hydrolases and other proteins targeted to lysosomes? I-cell disease helped decipher the signal for targeting proteins to the lysosome • Recessive mutation in single gene… • Fibroblasts of patients contain large inclusions (I-cells)… • Lysosomes lack normal complement of acid hydrolases… • All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)… • Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6phosphate)… • Lysosomal proteins of I-cells lack M-6-P… • Lysosomal targeting signal resides in carbohydrate! Mannose-6-P targets proteins from Golgi to lysosome Cis Golgi Network (CGN) Addition of M6P RER Lysosomal hydrolase (precursor) Trans Golgi Network (TGN) Transport via clathrin-coated vesicles to… Lysosome Clathrin coat Uncoupling Mature (pH 5) hydrolase M6P receptor Removal of phosphate & proteolytic processing… M6P receptor recycling back to Golgi Addition of M6P to lysosomal enzymes in cis-Golgi M6P receptor in TGN directs transport of enzymes to lysosome via clathrincoated vesicles Patients with I-cell disease lack phosphotransferase needed for addition of M6-P to lysosomal proteins in fibroblasts… secreted… Protein targeting, revisited Protein targeting Secretion/membrane proteins Cytoplasm Signal sequence (hydrophobic a-helix) RER Vesicle targeting Default or signal? KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default or signal? (constituitive secretion) Plasma membrane See ECB figure 14-5 Transport M6P Lysosomes Retrieval Next lecture: endocytosis and clathrin coats Lecture 17 The pathways to the lysosome Phagocytosis Autophagy Endocytosis Endocytosis- The inward limb of membrane cycling Pinocytosis Clathrin coated vesicles Receptor-mediated endocytosis Three pathways to the lysosome Phagocytosis Endocytosis Autophagy ECB 15-35 Phagocytosis - “cell eating” Performed by specialized “phagocytes:” WBCs 1. “Phagocytosis” Pseudopods Bacterium A. Entrapment by pseudopods B. Engulfment: pseudopods fuse to internalize prey in phagosome… C. Digestion: phagosome fuses with lysosome Phagosome Vesicles w lysosomal enzymes Some bacteria have evolved to evade digestion in lysosomes, and live as intracellular parasites or pathogens… Lysosome Myxobacteria tuberculosis (tuberculosis)… Listeria monocytogenes (listeria)… Yersinia pestis (plague)… Where do vesicles with lysosomal contents come from? What is their address label? Autophagy (“Self-eating”); used to recycle worn-out organelles 1. “Phagocytosis” Bacterium Phagosome Vesicles w lysosomal enzymes Endoplasmic reticulum Worn out organelle engulfed by ER Worn out mitochondrion 2. “Autophagy” Autophagosome Lysosome Endocytosis: Pinocytosis (“cell drinking”) and “receptor-mediated” endocytosis 1. “Phagocytosis” Bacterium Phagosome Early endosome Late endosome 3. “Endocytosis” Vesicles w lysosomal enzymes Endocytotic vesicles Endoplasmic reticulum Lysosome Worn out mitochondrion Autophagosome 2. “Autophagy” Note that vesicles from TGN targeted to lysosome by M6P actually fuse with precursor vesicles/organelles to form lysosome Overview of “pinocytosis” (“bulk” or “fluid-phase” endocytosis) Fluid-phase endocytosis can be followed in live cells with fluorescent dyes Proteinaceous coat Coated pit As many as ~2500 coated vesicles/min (~2-3% of surface area)! ~ 1’: early endosome (pH~6)… ATP GTP GDP+Pi H+ ADP+Pi ATP Coated vesicle ADP+Pi ATP Uncoating (seconds)… H+ ~ 5’: late endosome (pH 5.5~6)… ADP+Pi ATP H+ Delivery of acid hydrolases from TGN… ~30’: Lysosome (pH<5)… Early endosome - late endosome - lysosome is a continuum ADP+Pi EM views - coated pit to coated vesicle Coated pits ECB 15-18 coated vesicles Protein coat is “geodesic” clathrin cage Clathrin “heavy chain” “Light chain” 3 clathrin “heavy chains” (~180-190 kDa)… …plus… 3 clathrin light chains (~40 kDa)… …form… “Triskelions”… Spontaneously assemble into “geodesic” vesicle coats… 15.8-clathrin.mov Components of a clathrin-coated vesicle “cargo” Cargo and receptors we know from COP-coated vesicles receptors clathrin ECB 15-19 membrane adaptins Adaptins - adaptors that bind clathrin and cargo receptor, thereby regulating which cargo gets loaded into clathrin-coated vesicle Pinching off of vesicles requires the protein dynamin ECB 15-19 Coated pit budding “pinching off” uncoating (dynamin) Assembly of coat causes pit to form due to 3D shape of clathrin coat Dynamin is a GTPase ECB 15-19 GTP dynamin GTPase that regulates pinching off GDP Explains why non-hydrolyzable GTP analogues block endocytosis Clathrin-coated vesicles are rapidly uncoated Dynamin “Clathrin-coated pit” Adaptin complexes Clathrin By the “clathrin-uncoating ATPase” a member of the HSP70 family of chaperones; requires ATP hydrolysis GTP GDP + Pi Naked transport vesicles targeted to endosome… Clathrin and adaptins recycled Clathrin uncoating ATPase Naked transport vesicle See ECB figure 15-19 To endosome… ATP ADP + Pi Lecture 17 Phagocytosis Autophagy Endocytosis Endocytosis Pinocytosis Clathrin coated vesicles Receptor-mediated endocytosis How do cells take up specific macromolecules? “Receptor-mediated endocytosis” Example: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodies Lipid micelle: ~800 phospholipids… ~500 molecules of cholesterol… ~1500 molecules of cholesterol ester 1 copy of apoprotein B… Total mass: ~ 3 x 106 Da Overview of receptor-mediated uptake of LDL ECB 15-32 Low pH of endosome (~6) causes LDL to dissociate from receptor LDL is transferred to lysosome (fusion of vesicles from TGN) Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm for continued membrane biosynthesis in smooth ER Receptor is recycled back to surface (cycles about every 10 min!) Defects in LDL endocytosis are associated with “familial hypercholesterolemia”… –Severe atherosclerosis at early age (strokes and heart attacks in pre-teens) –Excess LDL in circulating blood –LDL not properly internalized by cells –Recessive/single gene… encoding plasma membrane receptor for LDL (LDL-receptor or LDL-R) •Disease provided insight into mechanism of receptormediated endocytosis and identification/function of LDL-receptor –Mutations in N-terminal domain: LDL-R doesn’t bind LDL… –Mutations in C-terminal domain: LDL-R is not internalized… What does this tell you about function of domains of LDL receptor? Domains in LDL receptor Based on MBoC (3) figure 13-53 LDL NH2 LDL-R N terminus of LDL receptor binds apoprotein B in LDL Plasma membrane HOOC Tyr Asn Val Pro Adaptin complex (four polypeptides) C terminus binds adaptin Recruitment of LDL-R to coated pits requires an “endocytosis signal” in cytoplasmic domain Based on MBoC (3) figure 13-53 LDL LDL-R Adaptin complex binds endocytosis signal in cytoplasmic domain of receptor: -NPXY- (Asn-Pro-Val-Tyr) in LDL-R Plasma membrane HOOC Tyr Asn Val Pro Endocytosis signal At least three different adaptin complexes; bind different endocytosis signals on receptors Adaptins recruit clathrin and initiate coated pit/vesicle formation Adaptin complex (four polypeptides) A single coated pit has many different receptors and cargos Low density lipoprotein (LDL) LDL-R 1,000s of receptors of many types per coated pit… Same coated pits used for pinocytosis! Summary of “receptor-mediated” endocytosis of LDL A single receptor makes hundreds of trips (~10 min/cycle) Low density lipoprotein (LDL) pH ~7-.7.2 LDL-R dynamin GTP GDP+Pi Early endosome ATP Coated vesicle pH ~6 ADP+Pi Uncoating (HSP70 family) ADP+Pi Fusion (Snares) ATP H+ Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptor Cholesterol ester cleaved Cholesterol released for use pH ~7.2 Free cholesterol Lysosome Coats for all reasons: a summary of vesicle coats and functions COPs: Outbound: ER to Golgi transport, intra-Golgi, Golgi to plasma membrane Retrograde: intra-Golgi, Golgi to ER Endosomal: early to late/lysosome Clathrin: Don’t worry about COPI vs II Plasma membrane to early endosome (endocytosis) Golgi to late endosome/lysosome Endosomes sort internalized receptors and ligands ECB 15-33 Some ligands Many receptors Many ligands Some receptors Maternal IgG –Secreted IgA –Others Transcytosis - movement of receptor to a different membrane from the one in which it was endocytosed “Transcytosis” moves maternal IgG across epithelia Intestinal lumen IgG in milk Milk duct IgG is “secreted” across the mammary epithelium into milk by transcytosis Apical membrane IgG receptor Receptor-mediated Tight junctions endocytosis from basolateral domain… Apical Endosome Secretion from apical membrane domain… IgG is transcytosed into the neonate blood Basolateral Endosome Endocytosis from apical domain and secretion to basolateral membrane Epithelial cell IgG in blood IgG receptor Basolateral membrane Neonate blood Maternal blood Polarized epithelial cells have distinct apical and basolateral endosome compartments Protein targeting and trafficking, finale! Secretion/membrane proteins Nucleus Protein targeting Signal peptide NLS: (basic) NES: (L-rich) Cytoplasm Signal sequence Peroxisomes SKL at C term. Transit peptide Mitochondria ECB considers these all to be signal sequences Chloroplasts Vesicle targeting RER KDEL (soluble proteins) KKXX (membrane proteins) Golgi Secretory vesicles (regulated secretion) Default M6P (constituitive secretion) Plasma membrane Transport Endosomes Endocytosis signal Lysosomes Retrieval Endocytosis: From plasma membrane to endosome to lysosome… Membrane flow during exocytosis and endocytosis is a delicate balance Golgi apparatus Endosome ER Lysosome Original surface Endocytosis internalizes membrane ~2-3% per minute… Block endocytosis, exocytosis continues: membrane area grows… Entire membrane is recycled in less than 1 hr… Block exocytosis, endocytosis continues: membrane area shrinks… Next lecture… “Cytoskeleton” Intermediate filaments: Cell structure Microfilaments: Muscle Organelle transport in plants Microtubules: Cilia and flagella Organelle transport in animals ECB 1-20