BioMG 1350: Week 7 Notes
BioMG 1350: Week 7 Notes Bio 1350
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This 38 page Class Notes was uploaded by Jheel Shah on Friday March 11, 2016. The Class Notes belongs to Bio 1350 at Cornell University taught by Dr. Tony Bretscher, Dr. Maria Garcia-Garcia in Spring 2016. Since its upload, it has received 83 views. For similar materials see Principles of Cell and Developmental Biology in Biology at Cornell University.
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Date Created: 03/11/16
3/6/16 Secretory Pathway Reading ECB3: 510-521 ECB4 503-515 Learning Objectives: • Understand the destinations of proteins synthesized on the ER • glycosylation occurs on proteins in the ER and is then modified in the Golgi • Explain what is meant by ER ‘quality control’ and how this is • Understand the topology and mechanism of vesicular transport between membrane- bound compartments, including the roles of coat proteins, • Understand how the Golgi apparatus functions in protein sorting t ie n ffic Last Lecture…………… S u n d ry a ss a e ce N Review of targeting to the ER, SRP and translocation: Movie 15-4 1 3/6/16 Roadmap of vesicle traffic Secretory pathway Figure 15-18 Essential Cell Biology The Endoplasmic Reticulum (ER) is a network of membranes spread throughout the cell. Fluorescence micrograph of EM: Specialized secretory cell ER at edge of cultured cell 2 3/6/16 The ER is the site of synthesis for membrane and lumenal proteins of the ER, Golgi, endosomes, lysosomes, and of secreted and plasma membrane proteins. (SRP and SRP receptor not shown) Golgi Apparatus Endosome Lysosomal Plasma membrane Proteins and secretory proteins Roadmap of vesicle traffic Secretory pathway Endocytic pathway Figure 15-18 Essential Cell Biology 3 3/6/16 Today’s topics 1. Events in the ER: Protein glycosylation, protein folding, protein Quality Control, and the Unfolded Protein Response Pathway 2. Transport by vesicles to the Golgi Apparatus: COPII 3. Protein modification in the Golgi, and transport out of it. In the active learning sections starting on Wednesday of this week, you will be examining the dynamics of a membrane protein synthesized on the endoplasmic reticulum and its transport to and within the Golgi Apparatus. Many Proteins are Glycosylated on Asparagine in the ER: ‘N’-linked glycosylation Called ‘N’-linked because N is (mRNA and the single letter Ribosome not code for shown) asparagine NEVER occurs Functions of N-linked glycosylation: in the cytosol • Helps protein folding and solubility • In some cases helps protein sorting • Protects protein in harsh environments • Can participate in biological function of ONLY ever occurs protein, eg, some cell in the ER lumen surface receptors ……..Asn-X-Ser/Thr……..(where X is any amino acid except proline) Figure 15-23 Essential Cell Biology 4 3/6/16 Chaperones assist protein folding and prevent misfolded proteins from leaving the ER S- S - S - S H - S H - S Chaperone: a protein that helps other proteins to fold correctly. Correct protein folding often involves disulfide bond formation between appropriate cysteine residues. This is possible because the ER is not a reducing environment. If the chaperone machinery cannot fold a protein properly, the protein is eventually degraded. This is called the ‘ER Quality Control System.’ Figure 15-24 Essential Cell Biology CFTR: Cystic Fibrosis TransmembraneConductance Regulator Location of Cl channel in unaffected individuals CFTR: Cystic Fibrosis TransmembraneConductance Regulator – it is a Cl - channel normally found in the plasma membrane Figure 15-18 Essential Cell Biology 5 3/6/16 The disease cystic fibrosis is due to a mutation in the gene for CFTR that causes the retention and degradation of the CFTR protein in the ER by the Quality Control System Mutant CFTR does not leave the ER and is degraded by the Quality Control System - CFTR: Cystic Fibrosis TransmembraneConductance Regulator – it is a Cl channel normally found in the plasma membrane Figure 15-18 Essential Cell Biology The ER adjusts its protein folding capacity when there are elevated levels of misfolded proteins: The Unfolded Protein Response (UPR) Figure 15-25 Essential Cell Biology 6 3/6/16 Today’s topics 1. Events in the ER: Protein glycosylation, protein folding, protein Quality Control, and the Unfolded Protein Response Pathway 2. Transport by vesicles to the Golgi Apparatus: COPII 3. Protein modification in the Golgi, and transport out of it. The Secretory Pathway and Protein Sorting Nucleus ER ‘resident’ protein Protein to be secreted out of cellelective transport Protein to be delivered to the lysosome by vesicles 7 3/6/16 Nuclear envelope Rough ER Transport vesicles Golgi Transport vesicles 1µm Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008) Vesicle budding and fusion: General Principles The asymmetry of the lipid bilayer is preserved, as is the orientation of membrane proteins The topology (ie orientation) of the lipid bilayer and membrane proteins is preserved Figure 13-2 Molecular Biology of the Cell 8 3/6/16 Vesicle budding and fusion with the plasma membrane The topology of the lipid bilayer and membrane proteins is preserved Figure 13-2 Molecular Biology of the Cell Vesicular transport maintains membrane topology Endoplasmic The topology of the Reticulum lipid bilayer and membrane proteins is preserved Golgi Apparatus Plasma Membrane The lumen of the ER, transport vesicles, and the Golgi correspond topologically to the ‘outside’ of the cell The regions of proteins with N-linked glycosylation are topologically on the lumenal side and therefore on the ‘outside’ of the cell Figure 13-2 Molecular Biology of the Cell 9 3/6/16 Roadmap of vesicle traffic Secretory pathway COPII transport vesicles Figure 15-18 Essential Cell Biology COPII vesicles transport selective cargo from the ER to the Golgi 1. Selection of cargo to be transported is mediated by transport receptors COPII =Coat Protein II 2. Adaptors capture cargo receptors by binding their cytoplasmic tails 3. Coat proteins bind adaptors to form a vesicle 4. Vesicle pinches from membrane 5. Vesicle is uncoated and ready to fuse with target membrane Coated Vesicle Transport Adaptors COPII Coat Vesicle Cargo receptor Cytosol ER Membrane ER Lumen Very heavily adapted from: Figure 15-20 Essential Cell Biology 10 3/6/16 Rab GTP Cycle and SNARES direct transport vesicles to their target membranes Dynein Rab-GEF v-SNARE = vesicle SNARE GDP t-SNARE = target SNARE Rab-GTP GTP Rab-GDP Microtubule Rab-GAP Modified from Figure 15-21 Essential Cell Biology SNARES play a central role in membrane fusion v- and t-SNAREs dissociate and v-snare recycled by endocytosis Figure 15-22 Essential Cell Biology 11 3/6/16 General Key Points about transport by membrane vesicles Membrane vesicles mediate transport between many different membrane-bound compartments. How do they form and know which compartment to fuse with? • Transport between membrane bound compartments involves transport vesicles. • Selection of cargo to be transported is mediated by receptors • Adaptors link the receptors to coat proteins • Coat proteins shape the transport vesicle • A Rab protein with bound GTP associates with the transport vesicle • The transport vesicle is docked through the Rab-GTP to tethering factors • Hydrolysis of Rab-GTP to Rab-GDP and Pi is activated by the Rab-GAP • The Rab-GDP protein leaves and is recycled by the Rab-GEF protein • A transport vesicle includes a v-SNARE as an identity tag • The target membrane has a t-SNARE as an identity tag • Membrane fusion is driven by tight association of the v-SNARE and t-SNAREs • There are specific Rab proteins and v-SNARE/t-SNAREs for each transport step Figure 13-2 Molecular Biology of the Cell Today’s topics 1. Events in the ER: Protein glycosylation, protein folding, protein Quality Control, and the Unfolded Protein Response Pathway 2. Transport by vesicles to the Golgi Apparatus: COPII 3. Protein modification in the Golgi, and transport out of it. 12 3/6/16 The Golgi apparatus is made of a stack of flattened, membrane-enclosed sacs called cisterna endoplasmic reticulum Functions of the Golgi Apparatus: 1. Sequential modification of the oligosaccharide chains. 2. Protein Sorting in the trans Golgi Network (TGN) Remember: This is true for both water-soluble lumenal secretory proteins, and proteins embedded in plasma lysosome the membrane. membrane via endosome Figure 15-26 Essential Cell Biology Oligosaccharide Modification Enzymes are found in distinct compartments of the Golgi Apparatus Unstained section Osmium Stain Nucleoside diphosphatase Acid phosphatase Figure 13-27 Molecular Biology of the Cell (© Garland Science 2008) 13 3/6/16 Secretory components are sorted in the TGN for the constitutive and regulated exocytic pathways Figure 15-27 Essential Cell Biology Figure 15-28 Essential Cell Biology (© Garland Science 2010) 14 3/6/16 The Membrane Traffic Sorting Protein synthesis on rough endoplasmic reticulum. N-linked glycosylation Quality control Modification, sorting and transport out of the Golgi complex Nucleus ConstitutivTransport, delivery and fusion at appropriate Regulatd sec destination reton Selective transport to the Golgi complex by COPII trans Golgi network vesicles cis medial trans ‘TGN’ 15 3/6/16 Membrane bound organelles are organized and transported along microtubules by ‘molecular motors’ Tvesiclet Readings for Next Lecture ECB3: 522-526 ECB4: 515-520 16 3/6/16 Lecture 10 Summary (page 1 of 3) • The endoplasmic reticulum is the site of synthesis of proteins of the ‘secretory’ pathway. This includes membrane proteins that will eventually either remain in the ER or be targeted to the Golgi, or endosomes, or lysosomes or the plasma membrane. It also includes proteins that are secreted out of the cell, or reside in the lumen of the ER, or Golgi, or endosomes or lysosomes. • During synthesis of proteins on the ER, whenever the sequence Asn-X-Ser/Thr comes through the translocon, a large oligosaccharide is transferred from a lipid-oligosaccharide donor to the asparagine side chain. This is called N-linked glycosylation. It never occurs on proteins in the cytosol. • Proteins have to be correctly folded in the ER. This often involves disulfide bond formation between appropriately located cysteine residues. If they are not properly folded, they are assisted in their folding by proteins known as chaperones. If they cannot achieve the correct folded state, they are removed and degraded by the quality control system. When properly folded, they may in incorporated into transport vesicles destined for the Golgi. • The level of chaperones in the ER is regulated so that when there are too many unfolded proteins, receptors in the ER sense them which induces the synthesis of more chaperones. This is know as the Unfolded Protein Response (UPR). • The cystic fibrosis transmembrane conductance regulator (CTFR) is a regulated plasma membrane Cl channel. A common mutation results in retention of the mutant CFTR in the ER by the quality control system and degradation of the mutant CFTR. • The pathway of secretory proteins was first discovered by following the itinerary of proteins synthesized on the endoplasmic reticulum. Proteins destined for the cell surface are synthesized on the ER, transported to the Golgi apparatus, where they undergo appropriate sequential modification of their oligosaccharide chains, and then packaged in vesicles and transported from the Trans-Golgi Network to the plasma membrane and released from the cell. • The endocytic pathway brings molecules in from outside the cell, or from the plasma membrane. • The secretory pathway and endocytic pathways intersect in endosomes. Lecture 10 Summary (page 2 of 3) • Transport between sequential membrane bound compartments involves the formation of a transport vesicle by budding, followed by fusion with the correct target membrane. During this process, both the membrane topology of the lipid bilayer is preserved, as is the topology of membrane proteins. Therefore the lumen of the ER, Golgi and endosomes are topologically equivalent to outside the cell. • COPII vesicles transport cargo from the ER to the Golgi apparatus. This involves capture of cargo by receptor proteins, binding of adaptors to link the receptors to the coat, the formation of a COPII coat to deform the membrane into a vesicle, the pinching off of the vesicles, subsequent shedding of the membrane coat, and fusion with the appropriate target membrane. • Transport vesicles need to have recognition markers on them so that they fuse with the correct target membrane. • Two major types of recognition markers are involved in each transport step: Rabs and SNAREs • Rabs are small GTP-binding proteins (‘molecular switches’) that in their active GTP-bound state associate with the transport vesicle. The active Rab on the vesicle binds to tethering factors on the target membrane, which docks the vesicle there. During this process, the GTP is hydrolyzed, and the Rab protein is recycled for another round of bringing in vesicles. There are many Rab proteins; each one is specific for a specific vesicular transport step. • Transport vesicles also carry a specific transmembrane protein called a v-SNARE. The v- SNARE binds to a specific membrane protein on the target membrane called a t-SNARE. There are many v-SNAREs and t-SNAREs, each specific for each transport step. 17 3/6/16 Lecture 10 Summary (page 3 of 3) • Binding of a v-SNARE to a t-SNARE brings the two lipid bilayers very close and drives membrane fusion. • There is a specific Rab and v-SNARE for each vesicular transport pathway. This ensures that vesicles bind and fuse with the correct target membrane. • Vesicles leaving the ER are targeted to the Golgi apparatus. The Golgi consists of a set of stacked membranes (like pita breads) called cisternae, each with distinct functions and through which secretory proteins are transported. The compartments are called cis-, medial- and trans-Golgi, and the final compartment is the trans-Golgi network (TGN). • The Golgi has two functions. First, the oligosaccharide side chains are sequentially modified as the proteins pass through the Golgi. Second, at the TGN they are sorted and transported in vesicles to their next compartment – such as the endosome on the way to the lysosome, or the plasma membrane. • There are two vesicular routes from the TGN that result in exocytosis at the plasma membrane. The ‘constitutive’ pathway delivers vesicles to the plasma membrane and is not regulated. Cargo whose release needs to be regulated – for example release of insulin following a hormonal signal – is first packaged into storage secretory vesicles and then released from the cell when the appropriate hormonal signal is received. This is called ‘regulated’ secretion. • All compartments of the secretory pathway – ER, Golgi, secretory vesicles – are localized and transported in the cell by microtubule-based motors on microtubules. 18 3/8/16 Endocytic Pathways Reading: ECB3 522-526 ECB4 515-520 Learning Objectives: • Understand that most vesicle trafficking pathways go in both directions, eg. COPII and COPI • Understand how the KDEL receptor ‘retrieves’ ER resident proteins • Understand principles of receptor mediated endocytosis, eg uptake of LDL • Understand function of lysosomes • Understand how lysosomal enzymes are selectively sorted to lysosomes • Understand how large particles are taken up by phagocytosis Last lecture 1 3/8/16 The Membrane Traffic Sorting Protein synthesis on rough endoplasmic reticulum. N-linked glycosylation Quality control Modification, sorting and transport out of the Golgi complex Nucleus Constitutive secretion Transport, delivery and fusion at appropriate Regultd destination sereton Selective transport to the Golgi complex by COPII trans Golgi network vesicles cis medial trans ‘TGN’ Today’s topics Finishing up the Secretory Pathway: 1. Retrieval pathway from the Golgi to the ER: COPI. Endocytic Pathways 1. The discovery of the LDL receptor mediated endocytic (RME) pathway 2. The role of Clathrin in vesicle formation 3. Lysosomes and their biogenesis 4. Phagocytosis 2 3/8/16 The Secretory pathway COPII Constitutive secretion Reguateds ecrton COPI Figure 15-18 Essential Cell Biology COPI transport vesicles mediate the retrieval of ‘escaped’ ER proteins COPI: Coat Protein I cis-Golgi Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008) 3 3/8/16 Soluble ER proteins contain the KDEL recognition signal, and bind the KDEL Receptor for transport back to the ER by COPI vesicles KDEL = lysine-aspartic acid-glutamic acid-leucine Figure 13-24a Molecular Biology of the Cell (© Garland Science 2008) COPI transport vesicles mediate the retrieval of ‘escaped’ ER proteins and recycling of v-SNAREs Active V-SNARE Inactive V-SNARE Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008) 4 3/8/16 How do the retrieval COPI vesicles know where to go? They have their own v-SNARES! Inactive retrieval v-SNARE Active forwards v-SNARE Inactive forwards Active retrievalv-SNARE v-SNARE Figure 13-24b Molecular Biology of the Cell (© Garland Science 2008) Distinct Membrane Coats are involved in transport vesicle formation from the ER to the Golgi (COPII) and back from the Golgi to the ER (COPI). Figure 13-4 Molecular Biology of the Cell (© Garland Science 2008) 5 3/8/16 COPII and COPI pathways COPII Constitutive secretion Reglatdse crtin COPI Adapters and coats define the composition of transport vesicles, and cargo transporters recycle. Figure 15-18 Essential Cell Biology Today’s topics Finishing up the Secretory Pathway: 1. Retrieval pathway from the Golgi to the ER: COPI Endocytic Pathways. 1. The discovery of the LDL receptor mediated endocytic (RME) pathway 2. The role of Clathrin in vesicle formation 3. Lysosomes and their biogenesis 4. Phagocytosis 6 3/8/16 Endocytic pathways Nucleus Constitutive secretion Regulted sereton Endocytosis: The taking in (‘uptake’) of material by the invagination of the plasma membrane. Modified from Fig. 15-18 Essential Cell Biology 3/e Cholesterol in the blood is carried by the LDL particle. Too much blood LDL causes hypercholesterolemia, which leads to atheroschlerosis and heart disease. High LDL is caused by genetic diseases called ‘familial hypercholesterolemias’ (FH) Nobel Prize 1985 LDL (Low density lipoprotein) FH is due to the inability to take up LDL from the blood 7 3/8/16 The LDL receptor on the cell surface binds LDL and internalizes it through clathrin coated pits Clathrin coated pit Cholesterol is carried by the LDL particle in the blood. Too much LDL causeh sypercholesterolaemia Mike Brown and Joe Goldstein showed that: • Normal cells have a high affinity receptor for LDL on their cell surface. • Some patients with very high levels of LDL suffer from hypercholesterolaemia because they have defective receptors that cannot bind LDL. Thus the receptor is critical for the uptake of LDL. • Other patients had cell surface receptors that bound LDL, yet could not take up LDL. The mutations were on the cytosolic part of the receptor. These receptors failed to localize to clathrin coated pits. • Localization of the receptor to coated pits must be necessary for the uptake of LDL. This process is now called receptor-mediated endocytosis (RME). X X 8 3/8/16 LDL uptake is an example of Receptor-Mediated Endocytosis (RME) early Low pH (about 6.0) The LDL receptor completes a cycle once every 10 minutes Many other proteins and particles have specific receptors and are taken up by RME. Figure 15-33 Essential Cell Biology (see movie 15.10) Endocytosis is exploited by many viruses to gain entry into cells For example: Flu virus HIV Polio virus Foot-and-mouth virus Hepatitis C virus 9 3/8/16 Today’s topics Finishing up the Secretory Pathway: 1. Retrieval pathway from the Golgi to the ER: COPI. Endocytic Pathways 1. The discovery of the LDL receptor mediated endocytic (RME) pathway 2. The role of Clathrin in vesicle formation 3. Lysosomes and their biogenesis 4. Phagocytosis 10 3/8/16 Adaptin-2 and cargo receptors select molecules for transport Notice: v-SNARE and Rab proteins are not shown! LDL -2 -2 Receptor LDL Modified from Figure 15-20 Essential Cell Biology Clathrin coated pits and vesicles View from inside the cell looking at the inner surface of the plasma membrane Figure 15-19b Essential Cell Biology 11 3/8/16 Clathrin movie Movie 15-5 Essential Cell Biology Integration of the Secretory and Endocytic Systems LDL LDL Receptor RME cycle Nucleus Constitutive secretion Reg uatedsecreti on Notice: Coats and adaptors not shown Modified from Fig. 15-18 Essential Cell Biology 3/e 12 3/8/16 Today’s topics Finishing up the Secretory Pathway: 1. Retrieval pathway from the Golgi to the ER: COPI. Endocytic Pathways 1. The discovery of the LDL receptor mediated endocytic (RME) pathway 2. The role of Clathrin in vesicle formation 3. Lysosomes and their biogenesis 4. Phagocytosis The lumen of lysosomes is acidic and full of hydrolytic enzymes • the proton pumpcidic due to the presence of • Lysosomes contain hydrolytic enzymes that can break down any biological molecule • They contain membrane transporters so that products of degradation can be reused • Many of the membrane proteins lining the lumen are heavily glycosylated to protect them from the harsh environment Lysosomalenzymes function optimally at pH 5.0, and hardly at all at pH 7.2. They are collectively called ‘acid hydrolases’. Their substrates are partially denatured at pH 5.0, so together this results in rapid substrate degradation. What do you think would happen if one of the lysosomal enzymes was missing? Fig. 15-35 Essential Cell Biology 3/e 13 3/8/16 The lumen of lysosomes is acidic and full of hydrolytic enzymes Some diseases associated with genetic defects in lysosomal enzymes: α-fucosidase (Fucosidosis) α-galactosidase (Fabry disease) α-iduronidase (Hurler syndrome; MPS I) α-mannosidase (α-mannosidosis) α-neuraminidase (sialidosis) β-galactosidase (GM1 gangliosidosis) β-glucosidase (Gaucher disease) β-glucuronidase (Sly syndrome; MPS VII) β-mannosidase (β-mannosidosis) Lysosomalenzymes function optimally at pH 5.0, and hardly at all at pH 7.2. They are collectively called ‘acid hydrolases’. Their substrates are partially denatured at pH 5.0, so together this results in rapid substrate degradation. How do lysosomal enzymes get sorted to lysosomes? Fig. 15-35 Essential Cell Biology 3/e Lysosomalenzymes are synthesized on the ER, transported to the Golgi and then selectively transported from the trans- Golgi Network to the early endosome and then on to the lysosome LDL LDL Receptor RME cycle Nucleus Constitutive secretion Regultedsec rton Modified from Fig. 15-18 Essential Cell Biology 3/e 14 3/8/16 Delivery of lysosomal enzymes (ie acid hydrolases) by recycling of the LysosomalEnzyme Receptor Binding to the lysosomal Enzyme Receptor MODIFICATION IN GOLGI Adaptin-1 and Heavily modified from: Integration of the Secretory and Endocytic Systems LDL LDL Receptor RME cycle Nucleus Constitutive secretion Regulaed sec reton Key: LDL Notice: Coats and adaptors not shown LDL Receptor Lysosomal enzyme Modified from Fig. 15-18 Essential Cell Biology 3/eomal enzyme receptor 15 3/8/16 Different Receptor Proteins can take Different Pathways Apical plasma membrane Basolateral plasma membrane Coated Vesicle Transport Pathways Type of Coated Vesicle Origin Destination COPII Endoplasmic Reticulum cis-Golgi COPI cis-Golgi Endoplasmic Reticulum Clathrin and Adaptin-1 Trans-Golgi Network Early Endosome Clathrin and Adaptin-2 Plasma Membrane Early endosome Retromer Early endosome Trans-Golgi Network 16 3/8/16 Today’s topics Finishing up the Secretory Pathway: 1. Retrieval pathway from the Golgi to the ER: COPI. Endocytic Pathways 1. The discovery of the LDL receptor mediated endocytic (RME) pathway 2. The role of Clathrin in vesicle formation 3. Lysosomes and their biogenesis 4. Phagocytosis Phagocytosis involves engulfing large particles (eg bacteria) and delivering them to the lysosome for degradation Phagocytosis involves: • engulfing the particle into a phagosome by enclosing it with the plasma membrane • transport and fusion with a lysosome where the lysosomal enzymes will break down the particle 17 3/8/16 Phagocytosis red blood cells macrophage Figure 15-32 Essential Cell Biology Readings for Next Lecture ECB3 609-624 ECB4 603-616 18 3/8/16 Lecture 11 Summary (page 1 of 3) • The secretory pathway involves synthesis of proteins on the ER, their transport to and processing in the Golgi apparatus, and then sorting and delivery to their final destination. • In addition to the ‘forward’ secretory pathway, there is also a ‘backward’ or ‘retrieval’ transport between compartments. For example, some soluble lumenal proteins normally remain in the ER. If they escape from the ER, they are recognized by their KDEL signal and captured in the Golgi by the KDEL receptor. This receptor with its cargo is packaged into COPI vesicles which are targeted back to the ER. The COPI coat proteins are different from the COPII coat proteins. • This COPI retrieval pathway also carries the forward v-SNAREs in an inactive conformation back to the ER. The COPI have their own active retrieval v-SNAREs, which are carried forward in an inactive state by COPII transport vesicles. • The endocytic pathway involves taking up material from outside the cell. Endocytic cargo is first delivered to the early endosome. • Work on the uptake of LDL (Low density lipoprotein, a particle containing protein, phospholipids and derivatized cholesterol and a major carrier of blood cholesterol) first established the receptor-mediated endocytic (RME) pathway. Critical to this study was use of human patients with very high levels of blood LDL due a defect in LDL uptake, and suffering from hypercholesterolemia. Many of these had defects in the LDL receptor, and this guided the discovery of the receptor. • This RME pathway involves: (a) binding of LDL to the surface LDL receptor; (b) internalization of the receptor-LDL complex from a coated pit into a coated vesicle; (c) delivery of the receptor- LDL complex to the early endosome; (d) under the mildly acidic conditions of the early endosome, the LDL and LDL receptor dissociate; (e) return of the LDL receptor to the cell surface; (f) movement of the free LDL from the early endosome to the late endosome, and then to the lysosome; (g) in the lysosome, the protein parts of LDL are degraded and the cholesterol released for use in membrane biosynthesis. Lecture 11 Summary (page 2 of 3) • Internalization of the LDL receptor in coated pits involves an interaction of the tail of the receptor with adaptin-2, which interacts with the coat protein, clathrin. Clathrin shapes the membrane into a vesicle, and the membrane then undergoes fission to generate a free vesicle. The vesicle then fuses with an early endosome. • Many different molecules are taken up by receptor mediated endocytosis. As this is one way to get into cells, many viruses use RME to gain entry and have evolved mechanisms using the acidic pH of the endosome to escape into the cytosol. • Lysosomes are organelles with a low lumen pH (about 5.0) because they have a proton pump in their membrane. Lysosomes contain many different hydrolytic enzymes, which are collectively called ‘acid hydrolases’ as they only function at this acidic pH. Lysosomes have enzymes to break down all biological macromolecules, including proteins, carbohydrates, nucleic acids and lipids. Defects in lysosomal enzymes cause diseases because defects in specific enzymes causes the substrates to accumulate that cannot be degraded. • Lysosomal enzymes are synthesized and subject to N-linked glycosylation in the ER, transported to the Golgi apparatus where their oligosaccharides chains are modified to identify them as enzymes destined for the lysosome. A specific receptor, the lysosomal enzyme receptor, in the trans-Golgi network collects them and, together with adaptin-1 and a clathrin coat, form a transport vesicle that goes to the early endosome. In the endosome the receptor/lysosomal enzyme separate due to its slightly acidic pH. The receptor is packaged into a retromer coated vesicle and returned to the trans-Golgi network, whereas the lysosomal enzyme moves to the lysosome. 19 3/8/16 Lecture 11 Summary (page 3 of 3) • Phagocytosis involves the engulfment of large particles, like bacteria, enclosing it with plasma membrane. It is then called a phagosome. The phagosome fuses with lysosomes to break down the contents of the particle. 20
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