B142.Protein Trafficking_ ER, Golgi, Secretory Vesicle, Exocytosis.pptx

Foundations of Medicine I Protein Trafficking Endoplasmic Reticulum, Vesicle Transport and Exocytosis  Zhaohui Xu, Ph.D. Department of Biological Chemistry 3220D MSRB III...

Foundations of Medicine I Protein Trafficking Endoplasmic Reticulum, Vesicle Transport and Exocytosis  Zhaohui Xu, Ph.D. Department of Biological Chemistry 3220D MSRB III [email protected] Dr. Xu has no industry-supported research or industry relationships to disclose. Notice to Students: Any distribution or posting of this content is prohibited without permission from the University of Michigan Medical School. For permission or questions reach out to [email protected] 2  Our goal is to use respectful, inclusive, nonbiased language at all times. We are committed to continuous improvement and welcome feedback. If applicable: We understand that some topics covered in this lecture can be emotionally difficult to process. Please remember that your class counselors are an excellent resource and available if you need them. Our Scientific Trunk leadership team is also available to offer support. 3  Learning objectives Endoplasmic Reticulum Discuss the role of Endoplasmic Reticulum in protein trafficking Describe the properties of signal sequences (signal peptides) Discuss the roles of the SRP, SRP receptor and the protein translocator (translocon) in identification, targeting and co-translational movement of growing polypeptide across ER membrane Describe how proteins are either translocated across or inserted into the ER membrane Discuss the importance of protein modification in the ER including N-linked glycosylation, disulfide bond formation, attachment of GPI anchor, and protein folding Discuss protein quality control in the ER, the fate of unfolded/misfolded proteins, especially the CFTR protein in cystic fibrosis 4  Learning objectives (continued) Vesicle Transport Describe the role of vesicle transport in intracellular protein trafficking Describe the key steps in vesicle budding, including the role of coat proteins, adaptor proteins, membrane-remodeling enzymes, and Arf GTPases Describe the key steps in vesicle fusion, including the role of Rab GTPases and SNARE proteins Describe the processes by which secretory and membrane proteins travel from ER to Golgi and from Golgi to ER (for retrieval of ER proteins) Describe the role of the Golgi apparatus in intracellular protein trafficking 5  Learning objectives (continued) Exocytosis Describe the protein sorting that occur in the Golgi Differentiate between constitutive and regulated secretion (exocytosis) Describe the maturation of secretory vesicles and their secreted products Describe the mechanism of synchronized release of neurotransmitter 6  Lecture outlines Part I. Endoplasmic reticulum a. Clathrin coat 1. The role of ER in protein trafficking 1. Vesicle fusion 2. Co-translational translocation a. Rab a. SRP b. SNARE b. Translocon 2. Vesicle transport between ER and Golgi c. Membrane protein biogenesis a. Recruitment of proteins into COPII 3. Protein modification in the ER vesicle a. Protein glycosylation b. The retrieval pathway b. Protein folding 3. Golgi c. Quality control (UPR, ERAD) d. Cystic fibrosis Part III. Exocytosis 4. Sorting in the TGN Part II. Vesicle transport 5. Constitutive secretory pathway 1. Vesicle budding a. Secretory vesicles 6. Synchronized release of synaptic vesicles 7  Topics directly relevant to medicine FACTOR X SANTO DOMINGO Cystic fibrosis and CFTRΔF508 Botulinum toxin Tetanus toxin 8 Protein trafficking in eukaryotic cells Protein synthesis occurs in the cytosol (by free ribosomes or ribosomes associated with the cytosolic face of ER membrane. Non-cytosolic proteins must be correctly trafficked to Various membranes (membrane proteins). Various membrane-bound organelles. Mitochondria, peroxisome, ER, Golgi, endosome, lysosome, nucleus Outside of the cell surface (secretory proteins). 9 Topologically equivalent spaces in eukaryotic cells Intracellular space, gray the cytosol the nucleus Extracellular space, orange outside the cell the lumens of organelles Protein trafficking between different families of intracellular spaces requires protein to move across biological membrane. 10 Three different mechanisms for protein trafficking Gated transport (from intracellular space to intracellular space) through nuclear pore complex between cytosol and nucleus, via nuclear pore complex Transmembrane transport (from intracellular space to extracellular space, aka protein translocation) through translocation pores from cytosol into the ER lumen from cytosol to mitochondria, peroxisome Vesicle transport (from extracellular space to extracellular space) via membrane-enclosed small vesicles secretory pathway (ER, Golgi, endosome, lysosome, cell surface) 11 Part I. Endoplasmic reticulum 12 Endoplasmic reticulum Endoplasmic reticulum (ER) is an extensive, net-like membrane-bound compartment in the cytoplasm. ER is organized into branching tubules and flattened sacs that are interconnected. ER membrane is continuous with the outer nuclear membrane. ER membrane encloses a single internal space, ER lumen, which is continuous with the perinuclear space. 13 The role of endoplasmic reticulum Rough ER: ER with ribosomes on its cytosolic surface. Involved in the synthesis of secreted proteins and membrane-bound proteins. Smooth ER: regions of ER not associated with ribosomes. Involved in lipid synthesis, Ca2+ storage, and detoxification 14 Rough ER is the entry point of protein secretory pathway Proteins destined for the ER as well as other organelles in the secretory pathway first enter ER. Golgi apparatus Endosomes Lysosomes Cell surface (membrane or secreted) Soluble proteins are released in the ER lumen. Membrane proteins are inserted into the ER membrane. 15  What is the signal for targeting the ER? The signal for import into the ER is the ER-targeting signal sequence: 14-24 amino acid sequence including one or more basic residues (positively charged) followed by a core of 8 or more hydrophobic amino acids. ER-targeting signal sequence is usually found at the N-terminus of a protein. These N-terminal signal sequences are cleaved inside the ER by signal peptidase (a proteolytic enzyme). 16 ER protein translocation is coupled to protein translation Translocation into the ER occurs while the rest of the protein is being synthesized by the ribosome (co-translational translocation). Ribosomes must attach to the cytosolic surface of the ER membrane before the first 70 or so amino acids are translated for translocation to occur. Proteins synthesized by free ribosomes in the cytosol cannot be translocated into the ER.  The SRP cycle 1. The emerging signal sequence in the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP). Protein translation is temporarily arrested. 2. The resulting complex is recruited to the ER membrane by the SRP receptor, in the vicinity of the protein translocator (translocon). 3. The RNC is released from SRP and attached to the translocon. Translation resumes and translocation starts. The The SRP SRP cycle cycle is is driven driven by by GTP GTP binding/hydrolysis binding/hydrolysis and and molecular molecular recognition. recognition. Both Both SRP SRP and and its its 4. SRP and its receptor dissociate receptor receptor are are G-proteins. G-proteins. from each other. Docking ribosome on translocon When the ribosome is properly docked on the translocon, the ribosome and the translocon form a tight seal. The peptide exit tunnel and the translocon form a continuous channel. As translation resumes, the polypeptide chain is transferred directly into and across the membrane through the translocon. Translocon is a protein translocation channel Translocon is also known as translocation channel or the Sec 61 complex (has three subunits α, β, γ). Translocon is gated both vertically and laterally. Vertical gate allows soluble protein to move into the ER lumen and is provided by a short plug of α-helix. Lateral gate allows the signal sequence and hydrophobic transmembrane segments to move into the lipid bilayer.  Translocation of soluble proteins into the ER lumen The positively charged residues in the ER signal sequence encounter the negatively charged phospholipids. The N-terminus is retained on the cytosolic side of the membrane due to the interaction. The following hydrophobic stretch of the signal sequence inserts and moves through the lateral gate into the lipid bilayer. The segment after the signal sequence moves through the translocon pore and into the ER lumen. Ribosome not shown for The signal sequence stays in the lipid layer simplification until it was cleaved off, whereas the rest of protein would be deposited into the ER lumen.  Membrane protein biogenesis Topology Topology of of aa membrane membrane protein protein means means The The number number ofof times times the the chain chain span span the the membrane. membrane. The The orientation orientation of of these these membrane-spanning membrane-spanning segment. segment. Integral membrane proteins located at the plasma membrane, membranes of intracellular compartments of the secretory/endocytic pathway are initially assembled at the ER. Inserted into the membrane co-translationally Final topology is determined Tertiary and quaternary structures are achieved  Insertion of transmembrane proteins into the ER membrane Type Type II transmembrane transmembrane protein protein aa single single transmembrane transmembrane segment segment aa cleavable cleavable N-terminal N-terminal signal signal sequence sequence aa N-terminal N-terminal lumenal lumenal domain domain and and aa C-terminal C-terminal cytosolic cytosolic domain domain The N-terminal signal sequence initiates co-translational translocation just like in a soluble protein. When the hydrophobic transmembrane segment enters the translocon, it stops transfer of the protein through the translocon. The translocon opens up laterally and both sequences move into the lipid bilayer. The signal sequence is cleaved and the transmembrane segment anchors the protein in the membrane. Protein synthesis continues at the cytosolic side till translation is completed.  Diseases related to ER translocation deficiency FACTOR X SANTO DOMINGO A rare inherited blood clotting disorder Deficiency of Factor X, a protein required for blood to clot DNA sequence analysis shows that there are no errors in the sequence of the normal secreted Factor X but there is a glycine to arginine mutation in the signal peptide sequence What could be a mechanism for this disorder?  Protein modifications in the ER and beyond For most proteins that enter the ER, their final destinations are beyond the ER. Regardless of their final destinations, most proteins achieve their native structures (folded and assembled) in the ER. Modification Locations Glycosylation ER lumen; Golgi lumen Disulfide bond formation ER lumen Protein folding/assembly ER lumen Proteolytic cleavage Golgi lumen and beyond  Protein glycosylation Major sites of modification Ser, Thr Asn Monosaccharide composition Simple Complex Processing organelles Golgi lumen ER lumen; Golgi lumen Function molecular recognition folding; stability; targeting; molecular recognition N-linked glycosylation in the ER The most common form of protein glycosylation in the ER N-linked modification by a preformed precursor oligosaccharide (14 sugars composed of 2 N-acetylglucosamine, 9 mannoses, and 3 glucoses). The assembly of the precursor occurs on a special lipid molecule dolichol anchored in the ER membrane. The precursor oligosaccharide is transferred from dolichol to the target asparagine in a single step by an oligosaccharyl transferase. Transfer occurs immediately after the target asparagine enter the ER lumen during protein translocation.  Attachment of GPI anchor in the ER Glycosyl phosphatidylinositol (GPI) anchor Phosphatidylinositol bears a short oligosaccharide covalently joined to the C- terminal residue of a protein through phosphoethanolamine. GPI anchored membrane proteins are initially inserted into the ER membrane like type I transmembrane proteins. A short sequence in the lumenal domain (adjacent to the transmembrane segment) is recognized by a GPI transamidase. The enzyme cleaves off the transmembrane segment and transfers the lumenal domain to a GPI anchor in the membrane. Disulfide bond formation in the ER Formation of Disulfide bridges ER is an oxidative environment that promotes the formation of S-S bridges. NOTE: S-S bonds are only formed in the ER. Protein Disulfide Isomerase (PDI) helps disulfide bond formation in the ER lumen. Oxidized PDI acts as an oxidant and facilitates the formation of S-S bonds in protein structure. Reduced PDI helps with the reformation of the correct disulfide bonds within the substrate.  ER-specific chaperones BiP (Hsp70) It assists various nascent and newly synthesized proteins to fold. It is involved in the unfolded protein response (UPR) and ER-associated degradation (ERAD). ERdj1–5 (Hsp40s) They contain a lumenally exposed J-domain that stimulates BiP ATPase activity. GRP94 (Hsp90) Calnexin and calreticulin ER lectins (carbohydrate-binding proteins) that assist the folding of proteins that carry certain N-linked glycosylation. Peptidyl-prolyl isomerases Catalyzes cis/trans isomerization of peptidyl-prolyl bonds.  Role of N-linked glycosylation in ER protein folding 3. 3. The The mono-glucosylated mono-glucosylated core core ligand ligand binds binds to to calnexin calnexin The or or calreticulin, calreticulin, which which serve serve as as molecular molecular chaperones, chaperones, calnexin/calrecticul preventing preventing aggregation aggregation and and export export of of the the incompletely incompletely in cycle folded folded chains chains from from the the ER. ER. 1. 1. A A core core glycan glycan is is 3 4 1 4. 4. To To release release the the bound bound added added to to the the growing, growing, proteins, proteins, glucosidase glucosidase nascent polypeptide nascent polypeptide 2 removes removes the the remaining remaining chain. chain. glucose residue. glucose residue. 2. 2. Two Two terminal terminal glucoses glucoses are are trimmed trimmed 5 off. off. 5. 5. Correctly Correctly folded folded 6. 6 glycoproteins glycoproteins are are now now 6. Incorrectly Incorrectly folded folded glycoproteins glycoproteins are are recognized recognized by by aa glucosyltransferase glucosyltransferase free free to to leave leave the the ER. ER. (GT), (GT), which which reglucosylates reglucosylates proteins. proteins. GT GT serves serves as as aa folding folding sensor sensor in in the the cycle. If reglucosylated, a glycoprotein cycle. If reglucosylated, a glycoprotein will will rebind rebind to to the the calnexin/calreticulum. calnexin/calreticulum. 31 The unfolded protein response (UPR) Proteins synthesized on the rough ER membrane cannot exit the ER until they are properly folded. Improperly folded proteins are retained in the ER by binding to molecular chaperones. The Unfolded-Protein Response (UPR) - Cell’s response to the accumulation of unfolded proteins in the ER. Transcriptional control: activation of genes to increase the protein-folding capacity of ER. Translational control: reduction of proteins entering the ER. Apoptotic control: If cells experience prolonged ER stress, they undergo apoptosis (programmed cell death). 32 ER associated degradation (ERAD) Proteins that cannot be properly folded are translocated back across the ER membrane and degraded in the cytosol by the proteasome. Misfolded proteins are recognized by chaperones and associated factors. They are targeted to the retro- protein translocator complex/E3 ubiquitin ligase. Translocated proteins are ubiquitylated (attachment of ubiquitin). Poly-ubiquitylated protein is targeted to the proteasome for degradation. 33 Cystic fibrosis Autosomal recessive genetic disorder caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene leading to defective versions of this protein. CFTR is a multi-spanning membrane protein (1480 amino acids) that functions as a Cl- channel found in apical membranes of exocrine epithelial cells that line passageways of the lungs, liver, pancreas, intestines, reproductive tract, and skin. 34  Cystic fibrosis One of the most common mutations that causes the disease, CFTRΔF508, result in a slightly unstable/misfolded form of CFTR that is retained in the ER and eventually degraded (even though the mutant protein will function normally if it can reach the plasma membrane). The quality control mechanism in the ER prevents the onward transport of misfolded or misassembled proteins that could potentially interfere with the function of normal proteins. A significant high percentage of newly synthesized polypeptides that enter the ER will never reach their final destinations. Accumulation of misfolded proteins can lead to unfolded protein response, ER- associated degradation and even apoptosis. 35 Part II. Vesicle transport 36 How does vesicle transport mediate protein trafficking? Vesicle transport - vesicles bud from membrane 1, transport, and fuse with membrane 2. Vesicle transport enables exchange of contents between different membrane-enclosed subcellular compartments without having to cross membranes. the lumens of ER, Golgi, endosome and lysosome as well as the extracellular space. the membranes of ER, Golgi, endosome and lysosome as well as the plasma membrane. 37 Intracellular protein trafficking pathways involving vesicle transport Biosynthetic-secretory Biosynthetic-secretory pathway pathway ER ER to to Golgi Golgi to to extracellular extracellular space space ER ER to to Golgi Golgi to to endosome endosome to to lysosome lysosome Retrieval Retrieval pathway pathway Endocytic Endocytic pathway pathway Extracellular Extracellular space space to to endosome endosome to to lysosome lysosome Autophagy Autophagy pathway pathway 38  Key steps in vesicle transport Transport vesicles form at specialized regions of membrane and bud off as coated vesicles. 39  Vesicles bud from coated regions of membranes Clathrin-coated Clathrin-coated vesicles vesicles From From Golgi Golgi to to endosome endosome From From plasma plasma membrane membrane to to early early endosome endosome COPI-coated COPI-coated vesicles vesicles From From Golgi Golgi back back to to ER ER COPII-coated COPII-coated vesicles vesicles From From ER ER to to Golgi Golgi Retromer-coated Retromer-coated vesicles vesicles The coat performs two major functions From From endosome endosome back back to to Golgi Golgi Helps select the appropriate molecules (cargoes) for transport. Deforms the membrane patch and molds the forming vesicles. 40  Clathrin-coated vesicles Structure of clathrin subunit: three heavy chains and three light chains form triskelion structure. These triskelions assemble to form basket-like framework of hexagons and pentagons. 41  Additional proteins required for coat assembly and disassembly Adaptor proteins recruit transmembrane Membrane-remodeling enzymes cargo receptors for specific cargo control the “pinching-off” of coated molecules. vesicles. form the inner layer of the coat between the Arf GTPases promote disassembly of clathrin cage and the membrane and anchor coats as soon as vesicles are budded the clathrin coat to the membrane. off from the donor membrane. 42 Rab GTPases guide vesicles to their target membrane Cells use Rab GTPases to ensure transport vesicles are targeted to the right membranes for fusion. Different organelles and membrane domains are marked with different Rab GTPases. Active Rab GTPase (GTP bound) interacts with its cognate Rab effector to establish the first connection between the two membranes that are going to fuse. 43 SNARE proteins mediate membrane fusion SNARE proteins are transmembrane proteins that catalyze the membrane fusion reactions in vesicle transport. SNARE proteins exist as complementary sets. v-SNAREs in vesicle membranes t-SNAREs in target membranes During fusion, a v-SNARE and a t-SNARE form a stable complex that locks the two membrane together. SNARE pairing provides an additional layer of specificity in vesicle fusion with their correct target membrane. 44 SNARE proteins form a stable helix bundle structure During fusion, water must be displaced from the hydrophilic surface of the membrane – energetically unfavorable. v-SNARE is a single polypeptide chain. t-SNARE is usually composed of three proteins. Both contain helical domains that are mostly unstructured in isolation. When they interact, the helical domain of one zipper up with the helical domains of the other to form a very stable four-helix bundle. The energy that is freed when the interacting helices wrap around each other is used to pull the two membrane faces together. 45 Recruitment of proteins into ER transport vesicles ER transport vesicles are COPII-coated. COPII-vesicles bud from ER exit sites (regions of ER without bound ribosomes). Protein entry into the vesicles can be a selective process (carrying an ER-specific exit signal recognized by adaptor proteins of COPII coat) or can happen by default (no signal). Proteins leaked out can be brought back later (retrieval pathway). 46 Some soluble proteins are recognized by cargo receptors Soluble cargo proteins exiting the ER are often mediated by transmembrane cargo receptors. Some of these cargo receptors are lectins that can recognize cargo proteins via N- linked glycans. Combined Combined factor factor V V and and factor factor VIII VIII deficiency: deficiency: LMAN1 LMAN1 (also (also known known as as ERGIC-53) ERGIC-53) is is aa mannose-specific mannose-specific lectin lectin in in the the ER. ER. LMAN1 LMAN1 and and MCFD2 MCFD2 form form aa protein protein complex complex andand serve serve asas aa cargo cargo receptor receptor toto transport transport coagulation coagulation factors factors V V and and VIII VIII from from the the ER. ER. Mutations Mutations inin either either LMAN1 LMAN1 oror MCFD2 MCFD2 cause cause this this rare rare bleeding bleeding disease. disease. 47 Vesicular tubular clusters mediate ER-Golgi trafficking ER transport vesicles are rapidly uncoated after budding. Uncoated vesicles fuse with each other to form vesicular tubular clusters (VTCs), which are intermediate structures between ER and Golgi and bring material from ER to Golgi. VTCs move quickly along microtubules to Golgi with which they will fuse. 48  The retrieval pathway from the Golgi back to the ER As soon as VTCs form, they begin to bud off transport vesicles of their own. These vesicles are COPI coated. They transport materials from VTCs (and from Golgi) back to ER as part of the retrieval pathway. What needs to be retrieved back to the ER? Cargo receptors that must be cycled from the Golgi back to the ER. ER resident proteins that are mistakenly packaged into the COPII vesicles. 49  ER retrieval signals The retrieval pathway depends on the ER retrieval signals. What are the ER retrieval signals? ER-resident soluble proteins: KDEL (luminal) Recognized by the KDEL receptor ER-resident membrane proteins (cytoplasmic) KKXX Di-arginine (XRRX) Why Why does does the the KDEL KDEL receptor receptor only only bind bind to to the the KDEL KDEL sequence sequence in in VTC VTC and and Golgi Golgi but but not not in in ER? ER? The The binding binding is is pH pH sensitive sensitive and and the the pH pH values values in in different different compartments compartments are are different different (lower (lower in in VTC VTC and and Golgi Golgi than than in in ER). ER). 50  The Golgi apparatus The Golgi apparatus consists of a collection of flattened, membrane- enclosed compartments. Cisterna: flattened, membrane- enclosed compartments. cis Golgi network (CGN): a collection of fused VTCs arriving from the ER. trans Golgi network (TGN) : exit site from the Golgi apparatus. 51  Golgi is the central hub in the protein trafficking pathway Modification of N-linked sugars (trimming of core mannose and addition of other oligosaccharides) and addition of O-linked sugars on Ser/Thr Mannose-6-phosphate addition on lysosomal proteins Sorting of proteins to lysosomes, secretory vesicles or plasma membrane (default) Sulfation of proteins and sugars 52 Part III. Exocytosis 53 Exocytosis and endocytosis Exocytosis and endocytosis are two ways for cells to exchange their cellular contents with their environment. Exocytosis - a transport vesicle fuses with the plasma membrane. Its content is released it into the extracellular space. Also known as secretion. Endocytosis - a plasma membrane patch is internalized, forming a transport vesicle. Its content derives from the extracellular space. 54 Constitutive and regulated secretory pathways Constitutive secretory pathway Provide new components for plasma membrane and extracellular matrix. Vesicles leave the TGN in a steady stream as irregularly shaped tubules. Occurs in all cells. Default pathway, no signal needed. Regulated secretory pathway Secret on-demand products (hormones, neurotransmitter, digestive enzymes) rapidly. Secretory proteins are initially stored in secretory vesicles and are released upon a signaling cue. Specialized secretory cells. Special signals required for packaging into the secretory vesicles. 55 Sorting proteins into secretory vesicles for regulated secretion Cells specialized for secretion stores their products in secretory vesicles. What is stored? Hormones, neurotransmitters etc. “Sorting signal”: proteins selectively aggregate with one another in TGN; signal patches Sorting machinery: not well understood Aggregation allow 200-fold concentration in secretory vesicles Exocytosis/secretion is triggered by a Mature secretory vesicles in the β-cells of the pancreas contains densely signaling cue (often a chemical signal). packed insulin molecules (secretory granules). 56 Maturation of secretory vesicles In immature secretory vesicles, membranes are loosely wrapped around the clusters of aggregated secretory proteins. Clathrin-coated vesicles return Golgi components back to the TGN (also allow cargo concentration) Secretory vesicles also fuse with one another and their lumens become progressively more acidic. 57  Proteolytic processing of secretory proteins Many secretory proteins (protein hormones, small neuropeptides, and secretory hydrolytic enzymes) are synthesized as inactive precursors. Proteolytic processing is needed to liberate them from their precursor forms. The cleavage often happens in the secretory vesicles. Several neuropeptides are synthesized as parts of a single polyprotein precursor. Proteolytic processing of the precursor occurs in secretory vesicles by cleavage at specific sites. Proteases for secreted proteins are active only in secretory vesicles (low pH). 58 Secretion of insulin Insulin is secreted by pancreatic β cells. Insulin is synthesized as proinsulin. Proinsulin is cleaved in secretory vesicles to release the mature insulin for secretion. Exocytosis of mature insulin aggregates from pancreatic β cells are stimulated by an external signal (e.g., by elevated blood glucose) 59  Secretion of neurotransmitters Synaptic vesicles: tiny secretory t-SNARE: syntaxin, SNAP-25; v-SNARE: synaptobrevin vesicles that store small Bacterial produced botulinum neurotoxin (causes neurotransmitter molecules. In botulism) and tetanus neurotoxin (causes tetanus) are response to Ca2+ influx, synaptic proteases that target neuronal SNARE proteins. vesicles can fuse with the plasma membrane in millisecond time scale. 60  Synchronized release of neurotransmitter 1. 1. Synaptic Synaptic vesicles vesicles are are 2. 2. In In the the primed primed state, state, docked docked at at the the the the SNAREs SNAREs are are partly partly presynaptic presynaptic plasma plasma paired paired butbut not not fully fully membrane. membrane. TheyThey assembled assembled into the into the undergo a priming undergo a priming final four-helix bundle. final four-helix bundle. step. step. 3. 3. Complexins Complexins freeze freeze 5. 5. The The SNARE SNARE bundle bundle the the SNARE SNARE complexes complexes zippers zippers up and aa fusion up and fusion in in the the primed primed state. state. pore opens. pore opens. 4. 4. When When intracellular intracellular [Ca [Ca ]] rises, 2+ 2+ rises, synaptotagmin synaptotagmin binds binds to to the the membrane membrane and and the the SNAREs, SNAREs, displacing displacing the complexins. the complexins. 61  Learning objectives Endoplasmic Reticulum Discuss the role of Endoplasmic Reticulum in protein trafficking Describe the properties of signal sequences (signal peptides) Discuss the roles of the SRP, SRP receptor and the protein translocator (translocon) in identification, targeting and co-translational movement of growing polypeptide across ER membrane Describe how proteins are either translocated across or inserted into the ER membrane Discuss the importance of protein modification in the ER including N-linked glycosylation, disulfide bond formation, attachment of GPI anchor, and protein folding Discuss protein quality control in the ER, the fate of unfolded/misfolded proteins, especially the CFTR protein in cystic fibrosis 62  Learning objectives (continued) Vesicle Transport Describe the role of vesicle transport in intracellular protein trafficking Describe the key steps in vesicle budding, including the role of coat proteins, adaptor proteins, membrane-remodeling enzymes, and Arf GTPases Describe the key steps in vesicle fusion, including the role of Rab GTPases and SNARE proteins Describe the processes by which secretory and membrane proteins travel from ER to Golgi and from Golgi to ER (for retrieval of ER proteins) Describe the role of the Golgi apparatus in intracellular protein trafficking 63  Learning objectives (continued) Exocytosis Describe the protein sorting that occur in the Golgi Differentiate between constitutive and regulated secretion (exocytosis) Describe the maturation of secretory vesicles and their secreted products Describe the mechanism of synchronized release of neurotransmitter 64