PHA114 Protein Sorting and Secretion PDF

WEEK 26 MPharm Programme Protein Sorting and Secretion Dr Matt Smith Slide 1 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Generation of proteins Slide 2 MPharm PHA114 Protein Sorting and...

WEEK 26 MPharm Programme Protein Sorting and Secretion Dr Matt Smith Slide 1 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Generation of proteins Slide 2 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Endocytosis (IN) and Exocytosis (OUT) Slide 3 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Where do proteins do their job? EVERYWHERE! Proteins are used in all parts of the cell: Nucleus Cytosol Organelles Cell membrane And outside the cell A very precise and regulated system is needed to move proteins into their site of action Slide 4 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Slide 5 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Overview of protein sorting mRNA is generated in the nucleus via transcription, mRNA is transported to the cytosol for translation on cytosolic ribosomes Non secretory pathway: 1. If the protein lacks an ER signal then translation is completed on free ribosomes in the cytosol (Non-secreted cytosolic proteins) 2. If the protein has an organelle-specific signaling sequence then it is generated in the cytosol and then targeted to its functional site (nucleus, mitochondria, peroxisome) (Steps 3-6: Slide 6) Secretory pathway (Also used to integrate proteins into membranes) 1. Proteins that are secreted are generated on cytosolic ribosomes and then target the ribosome to the ER (rough = ribosomes) 2. Translation is completed on the rough ER 3. The secretory proteins pass through to the golgi complex via transport vesicles 4. The golgi complex sorts proteins to the plasma membrane or to lysozymes Slide 6 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Protein secretion All eukaryotic cells use essentially the same secretory pathway for synthesizing and sorting secreted proteins and proteins that are found in the luminal space and in membranes Reasons for secretion: digestive enzymes, extracellular matrix, hormones, cell-to-cell signaling Three basic steps: Complex internal cellular architecture 1. Protein synthesis and allows proteins to move through the translocation across the ER membrane ER lumen 2. Protein folding and modification inside the ER lumen 3. Protein transport to the golgi, lysosomes or cell surface through budding and fusing of vesicles Slide 7 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Protein structure (more on this to come!) Proteins that need to be sorted into a specific place in the cell (not just in the cytosol) will have an N-terminal signal sequence that will direct the ribosome down to the endoplasmic reticulum One the ribosome has generated this sequence it will be directed to the ER via the SIGNAL RECONITION PARTICLE (SRP) The ER is responsible for sending out proteins to the correct place inside or outside the cell Slide 8 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Protein secretion 1. Signal sequence on mRNA  translated 2. Signal sequence bound by signal Co-translational translocation recognition particle (SRP), 3. SRP targets the ribosome to the SRP-receptor on the ER membrane (GTP required) 4. Opening of the translocon allows insertion of the signal sequence and the growing peptide chain. GTP hydrolysis – release of the signal sequence Slide 9 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Protein secretion 5. The polypeptide chain passes through the translocon and the signal sequence is cleaved by a Co-translational translocation membrane-bound peptidase enzyme. The signal sequence is then degraded in the ER lumen 6. The polypeptide is elongated in the N terminal to C-terminal direction – folding all the time. 7. The protein is released into the ER Lumen – ribosome is released into the cytosol Slide 10 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Common signal sequences Slide 11 MPharm PHA114 Protein Sorting and Secretion WEEK 26 The cell membrane Slide 12 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Inserting proteins into membranes Step 1. The ribosome (+ mRNA) attach to the translocon on the ER membrane 2. The protein is translated through the translocon until a hydrophobic stretch is generated and a membrane stop-transfer-anchor sequence is identified 3. The hydrophobic stretch is left in the membrane 4. The translocon moves laterally and ejects the protein 5. The rest of the protein protein is generated – this is external to the membrane 6. The complex dissociates Slide 13 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Inserting proteins into membranes Orientation of a protein in the membrane is established when it is first inserted into the membrane. This orientation of the protein persists all of the way to its final destination. That is, the cytosolic side of membrane remains on the cytosolic side throughout all processes. As membrane proteins are being translated, they are translocated or transferred into the ER until a hydrophobic membrane crossing domain is encountered. This serves as a 'stop transfer' signal and leaves the protein inserted in the ER membrane. Slide 14 MPharm PHA114 Protein Sorting and Secretion WEEK 26 The secretory pathway The pathway used to send soluble proteins and cell membrane bound proteins to be delivered to the: – Outside of the cell: e.g. Hormones, digestive enzymes – Into the plasma membrane e.g. receptors, channels – To lysosomes: e.g. acid tolerant digestive enzymes, H+ pumps Key point: this pathway is used to send proteins out of the cell or to membranes Slide 15 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Secretory Proteins 1. Newly generated proteins are translocated into the ER lumen for folding and glycosylation 2. These vesicles fuse with each other to form the cis-golgi. Proteins can be sent back to the ER (misfolded or ER resident proteins) via retrograde vesicles 3. Transport from the golgi to the ER is via retrograde vesicles 4. Transport through the golgi apparatus is via a non-vesicle process called cisternal maturation – restructuring of the gogi complex (movement of important enzymes) 5. Movement of golgi-resident proteins back to an earlier portion of the golgi is via retrograde vesicles Slide 16 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Secretory Proteins 6. Proteins that are secreted, are destined for the plasma membrane or lysosomes are sent to the trans-golgi network (TGN) 7. The TGN sends proteins to the cell membrane (via secretory vesicles) for regulated secretion 8. The TGN also sends proteins to the lysosomes 9. Vesicles coming into the cell are sent to the lysosome were the contents will be i) degraded; or ii) sent back via the TGN to the cell membrane Slide 17 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Vesicular trafficking The unifying principle of both the secretory and endocytic pathways is the use of membrane bound vesicles to move “cargo” between cell compartments Vesicles work by being able bud- off from one membrane and fuse with another membrane So the proteins only need to be A key point – proteins that translocated across a are integrated into a vesicle membrane once membrane keep the same orinetation to the cytosol True for both exocytosis and (inward facing or outward endocytosis facing) Slide 18 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Types of vesicle There are 3 types of vesicle: COP II: transport proteins from the rough ER to the golgi COP I: transport proteins in a retrograde direction between the golgi and the ER; and the different portions of the golgi Clathrin-coated: transport proteins from the plasma membrane to endosomes Slide 19 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Types of vesicle 1-3: Forward (anterograde) movement by COP II vesicles from ER to Golgi 4-6: Reverse (retrograde) movement by COP I vesicles from Golgi back to ER Not shown here - clathrin coated vesicles movement from the cell membrane into the cell (enodsome formation) Slide 20 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Molecular mechanisms of vesicle transport Vesicle budding: Budding is initiated by the recruitment of small GTP- binding proteins to the cell membrane causing an invagination Coat proteins in the cytosol then bind to cytosolic membrane cargo receptor proteins Cargo proteins are then recruited into the budding vesicle The membranes fuse and the vesicle is free The coat proteins are then lost and recycled Slide 21 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Molecular mechanisms of vesicle transport Vesicle docking: A vesicle will fuse to a target membrane via the interaction of SNARE proteins SNARE = SNAP Receptor proteins v-SNARE = vesicle SNARE t-SNARE = target membrane SNARE The SNARE proteins are in pairs and this ensures that the vesicle fuses with the correct Vesicle docking and unloading membrane SNARE: Soluble NSF attachment receptor protein NSF: N-ethylmaleimide-sensitive factor (Enzyme) – ATPase (you don’t need to know more than this) Slide 22 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Molecular mechanisms of vesicle transport COP II vesicle generation: ER to golgi Model of COPII coating of vesicles 1. A soluble GTP binding protein (Sar1) will interact with a membrane bound protein (Sec12) on the ER membrane – GDP is exchanged for GTP (activation). Activated Sar1 can then anchor into the membrane via a hydrophobic tail 2. Membrane bound Sar1 then acts as a binding site for coating proteins (Sec23/Sec24) and the budding of the vesicle 3. GTP hydrolysis = energy to remove the coat proteins 4. Vesicle is now free to fuse with the golgi Slide 23 MPharm PHA114 Protein Sorting and Secretion WEEK Docking 26 The docking of vesicles: Vesicle to plasma membrane: 1. A Rab protein (similar to Sar1 in function) integrated in the vesicle membrane binds to an effector protein on the plasma membrane outer surface = Targeting/Docking 2. A v-SNARE protein (vesicle: VAMP) interacts with its cognate t-SNARE complex (membrane: syntaxin and SNAP-25) 3. Stable docking – membrane fusion 4. NSF: NSF is a homohexameric ATPase that binds to the SNARE complex and through the hydrolysis of ATP causes the dissociation of the SNARE proteins SNAP: Synaptosomal-associated protein – allow specificity of vesicle docking VAMP: Vesicle-associated membrane protein SNARE: Soluble NSF attachment receptor protein Slide 24 MPharm PHA114 Protein Sorting and Secretion WEEK 26 The KDEL receptor: Retrieval of ER resident Proteins The retrieval of ER proteins from the golgi is carried out using the KDEL receptor. 1. ER luminal proteins are incorporated into COP II vesicles and transported to the golgi 2. Vesicle fusion at the golgi transfers the proteins into the golgi 3. ER resident proteins have a C-terminal KDEL signaling sequence (Lys-Asp-Glu- Leu). COP I vesicles transport these proteins back to the ER. The KDEL receptor is part of the vesicles (and golgi membrane) and binds proteins with the KDEL sequence. 4. The affinity of the KDEL receptor is higher at low pH (high H+) like in the golgi, but much lower at higher pH (lower H+) like in the ER Slide 25 MPharm PHA114 Protein Sorting and Secretion WEEK 26 The endocytic pathway Pathway used to bring proteins and other molecules into the cell across the plasma membrane Examples: – uptake of cholesterol and LDL particles – Iron atoms bound to transferrin proteins – Removal of receptor proteins from the cell surface Key point: this pathway is used to bring “stuff” into the cell Slide 26 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Clatherin-coated vesicle formation in endocytosis In order for molecules to be brought inside the cell a vesicle needs to be formed at the plasma membrane which internalises an extracellular (or membrane-bound) molecule An endocytic vesicle utilises several protein complexes: Dynamin: a GTPase Clatherin: fibrous protein (three-legged) Cargo proteins bind to specific receptors on the cell membrane causing the association of the clatherin complexes and invagination of the membrane Dymanin forms a spiral around the neck of the vesicle and uses GTP hydrolysis provide constriction energy. The vesicle then buds off the membrane into the cytosol. The cargo is then internalised to the vesicle Slide 27 MPharm PHA114 Protein Sorting and Secretion WEEK 26 The internalisation of low density lipoprotein (LDL) LDL: carriage of lipid, fat and cholesterol around the body – endocytic internalisation into cells via apoB protein 1. The cell surface has LDL receptors that bind to an apoB protein (in the LDL particle) – NPXY signal sequence used 2. Clatherin coated vesicles is formed and the LDL particle-receptor complex is internalised 3. Shedding of the coat proteins = early endosome (vesicle) fusion with late endosome – acidic pH of late endosome changes the conformation ApoB : Apolipoprotein (lipid transport) of the LDL receptor = release of the LDL particle 4. LDL breakdown receptor recycling Slide 28 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Protein modifications Membrane and soluble secretory proteins are: 1. Glycosylated (in the ER and Golgi) 2. Covalently stabilised by Cys-Cys bond formation (in the ER) 3. Assembled into multi-subunit conformations (in the ER) 4. Cleaved into their active conformation (in ER, Golgi and secretory vesicles) Slide 29 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Glycosylation of proteins Glycosylation of proteins = glycoproteins Carbohydrate chains are added to proteins via the: – Carboxyl groups of serine/threonine (O-linked) – Amide nitrogen of asparagine (N-linked) O-linked: 1 – 4 carbohydrate residues N-linked: Much more complex branched structures A pre-formed 14 residue N-linked glycan structure is added in the ER – 5 residues are conserved (always present) Slide 30 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Generation of N-linked core glycans Dolicol phosphate (DP) is a hydrophobic lipid (polyisoprenoid lipid) The first sugar (N-acetylglucosamine) is added to DP via pyrophosphate linkage The remaining sugars are added by glycosidic linkages (condensation reaction) The enzymes are found on the ER membrane A 7 residue intermediate is “flipped” across the ER membrane (cytosolic  ER lumen) The remaining manose and glucose residues are added via flipping DP with the residue attached across the ER membrane The 14-residue precursor is then transferred to an Asp residue in the ER lumen and trimmed Slide 31 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Generation of N-linked core glycans Dolicol phosphate (DP) is a hydrophobic lipid (polyisoprenoid lipid) The first sugar (N-acetylglucosamine) is added to DP via pyrophosphate linkage The remaining sugars are added by glycosidic linkages (condensation reaction) The enzymes are found on the ER membrane A 7 residue intermediate is “flipped” across the ER membrane (cytosolic  ER lumen) The remaining manose and glucose residues are added via flipping DP with the residue attached across the ER membrane The 14-residue precursor is then transferred to an Asp residue in the ER lumen and trimmed Slide 32 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Disulfide bond formation & re- arrangement Disulfide bonds are formed and re- arranged in the ER lumen Cys-Cys bonds stabilise protein structure Cys-Cys bonds are only found in secretory or membrane bound proteins Protein disulfide isomerase (PDI) carries out Cys-Cys bond formation Oxidised PDI is able to transiently bond with a protein and then form the Cys-Cys bond The oxidised PDI is Ero1 = Oxioreductin 1 (an oxidoreductase found in regenerated through the the ER lumen) activity of oxidised Ero1 Slide 33 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Chaperones help fold proteins Chaperones are proteins found in the ER lumen that help proteins fold into the native conformation (usually require ATP) They work by masking and stabilising exposed portions of the growing amino acid chain – usually stopping protein aggregation via hydrophobic interactions Slide 34 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Unfolded protein response The accumulation of unfolded proteins in the ER triggers the unfolded protein response The ER-membrane bound Ire1 protein has: ER luminal chaperone binding (BiP) activity Cytosolic RNA endonuclease activity When proteins are unfolded they cause the release of BiP chaperone proteins from the Ire1 protein (activating it) Activated Ire1 endonuclease activity cleaves un-spliced Hac1 mRNA which allows Hac1 protein translation Ire1 = inositol requiring endoribonuclease Hac1 = Transcriptional activator protein Hac1 then activates the expression of more chaperones and folding catalysts Slide 35 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Studying protein sorting and secretion Microscopy: Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged protein Pulse-chase experiments: 1. Cells are treated with [35S]methionine for 0.5h – this labels all newly synthesized proteins. The label is then removed and replaced with non-isotopic methionine 2. The proteins are then fractionated from the ER, Golgi and membrane 3. The proteins from each fraction are then visualized using autoradiography Slide 36 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Studying protein sorting and secretion Studying genetic secretory (sec) mutants: The secretory pathway is well characterized in terms of the protein effector molecules. Using genetic engineering and mutatgenesis one can study the effect of i) clinically relevant mutations and/or ii) therapeutic molecules on the secretory pathway Slide 37 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Summary The in vivo folding and packaging of proteins is a highly complex and highly regulated set of pathways As proteins are translated they are folded, modified and packaged for delivery to their site of activity The cell uses membrane bound vesicles to transport protein cargo into, out-of and internally round the cell Slide 38 MPharm PHA114 Protein Sorting and Secretion WEEK 26 Reference textbook Molecular Cell Biology (Lodish et al) – in the library… Slide 39 MPharm PHA114 Protein Sorting and Secretion