Cellular Organelles and Membrane Trafficking PDF

CHAPTER 12 Cellular Organelles and Membrane Trafficking Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.  Keys Emphasize the dynamic nature of the endomembrane system within the cell. Discriminate between regulated and...

CHAPTER 12 Cellular Organelles and Membrane Trafficking Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.  Keys Emphasize the dynamic nature of the endomembrane system within the cell. Discriminate between regulated and constitutive secretion. Elucidate the structure and function of the rough and smooth ER. Outline events in synthesis and transport of membranes/proteins through the cell. Elucidate role and sites of glycosylation in processing of secretory/integral membrane proteins. Elucidate the structure, function and polarization of the Golgi complex. Describe role of various types of coated- and non-coated-vesicles in membrane trafficking. Explain the signals used to target proteins to their appropriate cellular location. Describe the steps involved in the process of exocytosis and its triggers. Describe lysosomal structure/function and diseases caused by lysosome malfunction. Distinguish between phagocytosis, bulk phase endocytosis and receptor-mediated endocytosis. © 2013 John Wiley & Sons, Inc. All rights reserved.  Introduction Membranes divide the cytoplasm of eukaryotic cells into distinct compartments. The endomembrane system includes organelles such as the endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles functioning as part of a coordinated unit. © 2013 John Wiley & Sons, Inc. All rights reserved. (12.1) Overview of the Endomembrane System Membrane-bound compartments of Inside vesicle: orientation the cytoplasm remains the same Organelles of the endomembrane system are part of an integrated network in which materials are shuttled back and forth. Materials are shuttled between organelles in membrane-bound transport vesicles. Upon reaching their destination, the vesicles fuse with the membrane of the acceptor compartment. © 2013 John Wiley & Sons, Inc. All rights reserved.  Overview of the Endomembrane System Biosynthetic and secretory pathways Several distinct pathways through the cytoplasm have been identified. Biosynthetic pathway – Endocytic synthesis, modification and pathways that transport of proteins. unite Secretory pathway – when endomembranes proteins are discharged into a dynamic, (secreted) from the cell. interconnected network. – Constitutive secretion – in a continuous fashion. – Regulated secretion – in response to a stimulus. © 2013 John Wiley & Sons, Inc. All rights reserved. Overview of the Endomembrane System Synthesis and transport of secretory proteins 3 minute pulse: radioactively 3 minute pulse: 3 minute pulse: labeled amino acid (red) 17 minute chase 117 minute chase During regulated secretion, materials to be secreted are stored in large, membrane-bound secretory granules. – For example, secretion of digestive enzymes by pancreatic cells. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.2) Study of Cytomembranes Autoradiography Insights Gained from Autoradiography – Autoradiography – a method to visualize biochemical processes using radiolabeled materials exposed to a photographic film. – Can be used to determine where secretory proteins are synthesized by using labeled amino acids. – Techniques utilizing autoradiography have been largely supplanted by fluorescent-based approaches. Pancreatic acinar cell incubated 3 min with ‘hot’ amino acids. Silver grains localized over the ER © 2013 John Wiley & Sons, Inc. All rights reserved.  Study of Cytomembranes GFP-based protein tracking proteins can move Use of Green Fluorescent protein stuck in ER from ER to Golgi Protein – Green fluorescent protein (GFP) – a small protein isolated from jellyfish which emits green fluorescent light. – A GFP-DNA chimera allows to observe the protein synthesis in the cell. – Fusing viral genes to GFP allows the study The use of green fluorescent protein (GFP) reveals of protein traffic due the movement of proteins within a living cell. to large production of proteins. © 2013 John Wiley & Sons, Inc. All rights reserved.  Study of Cytomembranes Biochemical Analysis of Subcellular Fractions Smooth ER-derived Sub-cellular Fractions – Techniques to homogenize cells and isolate some organelles, which can then be separated from one another through sub- cellular fractionation. Rough ER-derived – Membrane vesicles derived from the endomembrane system form a collection of vesicles called microsomes, which can be characterized further through other Isolation of a techniques. microsomal fraction © 2013 John Wiley & Sons, Inc. All rights reserved.  Study of Cytomembranes Biochemical Analysis of Subcellular Fractions Use of Cell-Free Systems – Cell-free systems do not contain whole cells and have provided information about the roles of the proteins involved in membrane trafficking. – Other type of information identified includes: proteins that bind to the membrane to initiate vesicle formation, and proteins responsible for cargo Formation of coated vesicles selection. in a cell-free system © 2013 John Wiley & Sons, Inc. All rights reserved.  Study of Cytomembranes Utility of genetic mutants Study of Mutants – Mutants provide insights about the function of normal gene products. – Isolation of proteins from yeast has led to Use of genetic mutants in the study of secretion the identification of homologous proteins in mammals, pointing to the conserved nature of endomembrane systems. © 2013 John Wiley & Sons, Inc. All rights reserved.  Study of Cytomembranes Utility of genetic mutants RNAi reduces GFP-labeled secretory mannosidase II protein and becomes traps GFP- localized in the labeled Golgi complex mannosidase II in the ER Studying Mutants – RNA interference is a process in which cells produce small RNAs (siRNAs) that bind to specific mRNAs and inhibit the translation of these into proteins. – Scientists can identify genes involved in a particular process by determining which siRNAs interfere with that process. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.3) The Endoplasmic Reticulum The endoplasmic reticulum (ER) comprises a network of membranes that penetrates much of the cytoplasm. Like other organelles, the ER is highly dynamic undergoing continual turnover and reorganization. Electron micrograph: rough ER Divided into two subcompartments: – Rough endoplasmic reticulum (RER) & Smooth endoplasmic reticulum (SER) – In addition, the composition of the luminal or cisternal space inside ER membranes is different from the surrounding cytosolic space. Electron micrograph: smooth ER © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Schematic diagram RER: showing stacks of – Composed of a flattened cisternae network of flattened that make up the sacs (cistenae). rough ER – Continuous with the outer membrane of the nuclear envelope and also has ribosomes on its cytosolic surface. – Different types of cells have different ratios of the two types of ER, depending on activities of the cell. Transmission electron Immunofluorescence for micrograph of the rough ER the ER enzyme protein disulfide isomerase © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum SER – Extensively developed in a number of cell types; functions include: Synthesis of steroid hormones in endocrine cells: endocrine cells of the gonad and adrenal cortex. Detoxification in the liver of various organic compounds: home of the P450 enzymes. Sequestration of calcium ion from cytoplasm of Leydig cell: extensive SER where muscle cells: contains a steroid hormones are synthesized high concentration of calcium-binding proteins. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Polarized structure of a secretory cell, a mucus secreting goblet cell from rat colon. Low-power EM, mucus-secreting cell from Brunner's gland the mouse small intestine Functions of the RER – Polarity of RER in some cells reflects the flow from the site of protein synthesis to the site of discharge of the protein. – It is the starting point of the biosynthetic pathway. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Functions of the RER – Synthesis of Proteins on Membrane-Bound versus Free Ribosomes Approximately one-third polypeptides encoded by the human genome are synthesized on ribosomes of RER include secreted proteins, integral membrane proteins, and soluble proteins of organelles. Polypeptides synthesized on “free” ribosomes include: cytosolic proteins, peripheral membrane proteins, nuclear proteins, and proteins incorporated into chloroplasts, mitochondria, and peroxisomes. Site of synthesis of a protein determined by sequence of amino acids in N- terminus. – A signal sequence at N-terminus attaches to secretory proteins. – Polypeptide moves into ER cisternal space through a protein-lined pore. – Movement through the membrane can occur as it is being synthesized (co-translationally) or post-translational. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Synthesis of protein on membrane-bound ribosome A schematic model of the synthesis of a secretory protein (or a lysosomal enzyme) on a membrane-bound ribosome of the RER Synthesis of Secretory or Lysosomal protein on Membrane-Bound Ribosomes – Messenger RNA binds to free ribosomes on cytosol. – Secretory proteins synthesized on membrane-bound ribosomes have their signal sequence recognized by a signal recognition particle (SRP) © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Synthesis of protein on membrane-bound ribosome Cross-sectional view Ribosome of the translocon translocon channel from X-ray complex in the crystal structure act of protein synthesis Binding to the ER occurs through two sequential interactions: Initially, the SRP must interact with a SRP receptor. Then the ribosome interacts with the translocon, which is a protein-lined channel. Once the SRP-ribosome-nascent peptide chain complex binds ER, SRP is released. Release of SRP requires GTP-binding proteins (G proteins). © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Synthesis of protein on membrane-bound ribosome Processing of Newly Synthesized Proteins in the ER – Upon entering the RER lumen, the signal sequence is cleaved by a signal peptidase. – Carbohydrates are added by the enzyme oligosaccharyltransferase. – The RER lumen is packed with chaperones to assist in folding, and also contains protein disulfide isomerase to add disulfide bonds to cysteines. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Synthesis of protein on membrane-bound ribosome A schematic model for the synthesis of an integral membrane protein Synthesis of Integral Membrane Proteins on Membrane-Bound Ribosomes – Integral proteins contain hydrophobic trans-membrane segments that interfere with transfer into the RER lumen. – The translocon assists in the proper orientation of transmembrane sequences. – The arrangement within the membrane is determined by the orientation of the first trans-membrane segment is inserted. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Membrane Biosynthesis in the ER – Membranes arise from pre- existing membranes. – Lipids are inserted into existing membranes. – As the membrane moves one compartment to the next, its proteins and lipids are modified. – Membrane asymmetry is established initially and maintained during trafficking. Maintenance of membrane asymmetry © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Modifying the lipid composition of membranes Histogram indicating percentage of each of three phospholipids in three different cellular membranes Synthesis of Membrane Lipids – Most membrane lipids synthesized within the ER except sphingomyelin and glycolipids, and some unique lipids of mitochondria and chloroplasts. – Newly synthesized phospholipids are inserted into half of bilayer facing the cytosol, and then flipped into opposite leaflet by flippases. – There are enzymes that modify lipids already present within a membrane. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Modifying the lipid composition of membranes Contributing factors to variation of organelle lipid Schematic diagram contribution showing three – Organelle-specific distinct mechanisms: enzymes for lipid 1. Enzymatic conversion. modification (head group) – Inclusion/exclusion 2. Modification process during vesicle during vesicle formation. formation – Lipid-transfer proteins 3. Modification by that bind and transport phospholipid lipids without the use of transfer proteins vesicle transport. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Synthesis of the core portion of an oligosaccharide Glycosylation in the RER – Addition of sugars is catalyzed by glycosyltransferases. – Core segment of each carbohydrate chain is put together on a lipid carrier (dolichol phosphate) and then transferred to a polypeptide. – Core carbohydrate is modified by oligosaccharyltransferase as the polypeptide is transferred into the ER Steps in the synthesis of the core portion of lumen. an N-linked oligosaccharide in the rough ER © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Glycosylation in the RER – Soon after transfer to the polypeptide, the oligosaccharide is gradually modified. – A glycoprotein goes through a system of quality control to determine its fitness for a specific compartment. – Misfolded proteins are tagged by a terminal glucose and recognized by chaperones for refolding. – If the protein does not correctly fold, it is translocated to the cytosol and destroyed. Quality control: ensuring that misfolded proteins do not proceed forward. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Mechanisms that ensure the glucosyltransferase destruction of misfolded proteins – Misfolded proteins are not destroyed in the ER; instead they are transported into the cytosol where they are destroyed in proteasomes. – This process is called ER- associated degradation calreticulin (ERAD), and ensures the misfolded proteins do not reach the cell surface. Quality control: ensuring that misfolded proteins do not proceed forward. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Destruction of misfolded proteins – Accumulation of misfolded -chaperones proteins triggers the -transporters unfolded protein response -degradation (UPR). – Sensors in the ER are kept A model of the inactive by the chaperone mammalian BiP but if misfolded proteins unfolded protein are accumulated, BiP response (UPR). molecules are incapable of ATF6 inhibiting the sensors. PERK – Activated sensors send signals to trigger proteins involved in destruction of misfolded proteins. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endoplasmic Reticulum Visualizing membrane traffic with the use of a fluorescent tag: Movement of vesicular-tubular carriers: ER to Golgi with VSV-G:GFP tag From the ER to the Golgi Complex: The First Step in Vesicular Transport – RER have specialized exit sites where transport vesicles are formed (no ribosomes). – Transport vesicles fuse with one another and form the ERGIC (endoplasmic reticulum Golgi intermediate compartment) toward the Golgi complex. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.4) The Golgi Complex Schematic model of a portion of a EM: Golgi cisterna showing a Golgi complex from an epithelial cell concave central domain and an of the male rat reproductive tract. irregular peripheral domain The Golgi complex is a stack of flattened cisternae. It is divided into several functionally distinct compartments. The cis face of the Golgi faces the ER; the trans face is on the opposite side of the stack. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Golgi Complex Electron tomographic Fluorescence micrograph Electron micrograph image from a mouse of a cultured mammalian of a single isolated pancreatic beta cell cell towards COPI protein Golgi cisterna The Golgi complex is a stack of flattened cisternae. It is divided into several functionally distinct compartments. The cis face of the Golgi faces the ER; the trans face is on the opposite side of the stack. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Golgi Complex Regional differences in membrane composition across the Golgi stack: 1) Osmium tetroxide in the cis cisternae 2) Mannosidase II in the medial cisternae 3) Nucleotide diphosphatase in the trans cisternae The cis Golgi network (CGN) functions to sort proteins for the ER or the next Golgi station. The trans Golgi network functions in sorting proteins either to the membrane or various intracellular destinations. The Golgi complex is not uniform in composition; there are differences in composition from the cis to the trans face. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Golgi Complex Glycosylation steps of a typical mammalian N-linked oligosaccharide in the Golgi Glycosylation in the Golgi Complex – Assembly of carbohydrates found in glycolipids and glycoproteins takes place in the Golgi. – Sequence of incorporation of sugars into oligosaccharides is determined by glycosyltransferases. – Glycosylation steps can be diverse © 2013 John Wiley & Sons, Inc. All rights reserved.  The Golgi Complex VSV The dynamics of transport through the Golgi complex -mann II The Movement of Materials through the Golgi Complex – In the vesicular transport model, cargo is shuttled from the CGN to the TGN in vesicles. – In the cisternal maturation model, each cistern “matures” as it moves from the cis face to the trans face. – Current model: similar to cisternal maturation model but with vesicle retrograde transport. Golgi cisternae serve an primary anterograde carriers. © 2013 John Wiley & Sons, Inc. All rights reserved. (12.5) Types of Vesicle Transport and Their Functions Electron Electron micrograph: micrograph: COPII-coated COPI-coated vesicle vesicle Materials are carried between compartment using coated vesicles. Protein coats have two distinct functions: – Cause the membrane to curve and form a vesicle. – Select the components to be carried by vesicle. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Types of coated vesicles: – COPII-coated vesicles – move materials from the ER “forward” to the ERGIC and Golgi complex. – COPI-coated vesicles – move materials from ERGIC and Golgi “backward” to ER, or from the trans Golgi to the cis Golgi cisternae. – Clathrin-coated vesicles – move materials from the TGN to endosomes, lysosomes, and plant vacuoles. Proposed transport between membrane compartments of the biosynthetic- secretory pathway © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Proposed roles of the COPII coat proteins in generating membrane curvature, assembling the protein coat, and capturing cargo. COPII-Coated Vesicles: Transporting Cargo from the ER to the Golgi Complex – Proteins selected include enzymes and membrane proteins. – A small G protein called Sar1 plays a regulatory role in vesicle assembly. – Sar1-GTP binds to the ER. – Disassembly is triggered by hydrolysis of GTP which produces Sar1-GDP. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions A molecular model of the outer Sec13-Sec31 cage of the COPII coat as it would assemble around the surface of a 40-nm “vesicle.” COPII-Coated Vesicles: Transporting Cargo from the ER to the Golgi Complex – Proteins selected include enzymes and membrane proteins. – A small G protein called Sar1 plays a regulatory role in vesicle assembly. – Sar1-GTP binds to the ER. – Disassembly is triggered by hydrolysis of GTP which produces Sar1-GDP. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions COPI-Coated Vesicles: Transporting Escaped Proteins Back to the ER − Adenosylation ribose factor 1 (Arf1), a membrane-bending G protein, is required for vesicle transfer between cisternae. − Proteins are maintained in a organelle by two mechanisms: Retention of resident molecules that are excluded form transport vesicles. Retrieval of “escaped” molecules back to the compartment where they reside. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Resident proteins of the ER contain an amino acid sequence at the C-terminus serving as a retrieval signal. Specific receptors capture the molecules and bring them to the ER in COPI- coated vesicles. Each membrane compartment may have its own retrieval signals. Retrieving ER proteins: ER soluble proteins: KDEL ER membrane proteins: KKXX © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Beyond the Golgi Complex: Sorting Proteins at the TGN – Sorting and Transport of Lysosomal Enzymes Clatherin Lysosomal proteins are tagged with coated phosphorylated mannose residues. Tagged lysosomal enzymes are recognized and captured by mannose 6- phosphate receptors (MPRs). Targeting lysosomal enzymes to lysosomes: tagging with mannose 6-phosphate © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Sorting and Transport of Lysosomal Enzymes – Lysosomal enzymes are transported from the TGN in clathrin-coated vesicles. – The coats of these vesicles contain: Outer lattice composed of clathrin. Inner shell composed of protein adaptors. – Lysosomal enzymes are escorted from the TGN by Formation of clathrin-coated adaptor proteins GGAs. vesicles at the TGN © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Sorting and Transport of Non-lysosomal Proteins – Secretory proteins aggregate in dense granules that emerge form the TGN. – Plasma membrane proteins have different sorting signals in the cytoplasmic domain. – Polarized cells segregate apical membrane proteins and lateral/basal membrane proteins at the TGN into separate carriers. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Rab proteins on the vesicle and target membrane are involved in recruiting tethering proteins that mediate initial contact between the two membranes Synaptic vesicles associated with plasma membrane Targeting Vesicles to Particular Compartment - Tethering: Rab-mediated (G protein; < 60 in humans - Docking vesicles to target compartment. Protein that engage in these interactions are SNAREs (< 35 members.) SNAREs: v-SNAREs (incorporated into vesicles) and t-SNAREs (located in target). - Fusion: vesicle and target membranes. Interactions between t- and v-SNAREs pull the two lipid bilayers together. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Docking stage: a v-SNARE in the vesicle Synaptic membrane vesicle model interacts with showing the the t-SNAREs distribution of the SNARE synaptobrevin Targeting Vesicles to Particular Compartment - Tethering: Rab-mediated (G protein; < 60 in humans - Docking vesicles to target compartment. Protein that engage in these interactions are SNAREs (< 35 members.) SNAREs: v-SNAREs (incorporated into vesicles) and t-SNAREs (located in target). - Fusion: vesicle and target membranes. Interactions between t- and v-SNAREs pull the two lipid bilayers together. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Contributing proteins Model of interactions between  helices from syntaxin v- and t- SNAREs leading to (red), SNAP-25 (green), membrane fusion and and synaptobrevin exocytosis. (blue), with hypothetical 1) Synaptic vesicle docks to transmembrane helices the plasma membrane (yellow). through the formation of four-stranded protein SEM: surface of two bundles. alveolar (lung) cells 2) A speculative transition stimulated to discharge state in the fusion of the proteins stored in two membranes. secretory granules. 3) The transmembrane helices are now present in the same bilayer and a fusion pore has opened between the vesicle and target membrane. © 2013 John Wiley & Sons, Inc. All rights reserved. Types of Vesicle Transport and Their Functions Exocytosis – discharge of a secretory vesicle or granule after fusion with plasma membrane. – Process is triggered by an increase in [Ca2+]. – Contacts between vesicle and plasma membrane lead to formation “fusion pore”. – The luminal part of the vesicle membrane becomes the outer surface of the PM, and the cytosolic part becomes part of the inner surface of the PM. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.6) Lysosomes Lysosomes contain acid hydrolases which can digest every type of biological molecule. The low pH optimum of these enzymes is maintained by a proton pump (H+-ATPase) Portion of a phagocytic Kupffer cell of the liver showing at least 10 lysosomes of highly variable size © 2013 John Wiley & Sons, Inc. All rights reserved.  Lysosomes Autophagy Lysosomes play a key role in organelle turnover. During autophagy, an organelle is surrounded by a double membrane and a structure called an autophagosome is EM: a mitochondrion produced. and peroxisome The autophagosome is enclosed in a double membrane wrapper. then fused with a The autophagic vacuole lysosome to produce (autophagosome) would an autophagolysosome. have fused with a The digestive process A summary of the lysosome and its leaves a residual body. contents digested. autophagic pathway © 2013 John Wiley & Sons, Inc. All rights reserved.  The Human Perspective: Disorders Resulting from Defects in Lysosomal Function Lysosomal malfunctions can have serious effects on human health. Lysosomal storage disorders result from the absence of specific lysosomal enzymes thus allowing undigested material to accumulate. Lysosomal storage disorders can have a wide variety of symptoms. Among the best-studied disorders is Tay-Sachs disease. – It results from a deficiency in an enzyme responsible for degrading gangliosides, a major component of cell membranes. – It has a high incidence among Jews of eastern European ancestry. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Human Perspective: Disorders Resulting from Defects in Lysosomal Function © 2013 John Wiley & Sons, Inc. All rights reserved.  The Human Perspective: Disorders Resulting from Defects in Lysosomal Function Electron micrograph of a section through a portion of a neuron of a person with a lysosomal storage disease characterized by an inability to degrade GM2 gangliosides. Treatment for lysosomal storage diseases may include: – Enzyme replacement therapy – some treatments have either been approved or are being investigated – Substrate reduction therapy –currently in pre-clinical trials. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.7) Plant Cell Vacuoles Cylindrical leaf cells Transmission of the aquatic plant electron Elodea with a large micrograph of central vacuole a soybean surrounded by a cortical cell layer of cytoplasm showing the containing the large central chloroplasts vacuole A vacuole is a membrane-bound, fluid-filled compartment. Plant vacuoles have several storage functions. The vacuole membrane (tonoplast) contains an active transport system to keep a high concentration of ions so that water enters by osmosis. Plant vacuoles contain acid hydrolases. © 2013 John Wiley & Sons, Inc. All rights reserved.  (12.8) The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Endocytosis – uptake of cell surface receptors and bound extracellular ligands. Phagocytosis – uptake of particulate matter. Endocytosis can divided into two categories: – Pinocytosis – nonspecific uptake of extracellular fluids. – Receptor-mediated endocytosis – uptake of specific extracellular ligands following their binding to receptors. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Receptor-Mediated Endocytosis (RME) and the Role of Coated Pits – Substances that enter the cell through clathrin-mediated RME become bound to coated pits on the plasma membrane. – Clathrin-coated regions invaginate into the cytoplasm and then pinch free of the cytoplasm. Receptor-mediated endocytosis: Uptake of yolk lipoproteins by hen oocyte © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior LDL-cholesterol Clatherin-containing Cytosolic surface droplets on polygons on inner showing invagination extracellular surface surface of cell of clathrin coated pit. Receptor-mediated endocytosis – When viewed from the cytoplasm, coated pits appear as polygons resembling a honeycomb. – Geometric design is derived from structure of clathrin blocks. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Receptor-mediated endocytosis – Clathrin contains three heavy and three light chains that form a triskelion. – Coated vesicles also contain adaptors between clathrin and membrane. – One adaptor is EM of a metal-shadowed preparation of clathrin AP2. triskelions. Inset: triskelion composed of three heavy chains with inner portion linked to a smaller light chain © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Molecular organization of a coated vesicle Schematic showing Clathrin cage with interactions of the 36 triskelions Schematic of a coated vesicle surface AP2 adaptor with showing their showing arrangement of triskelions clathrin coat and overlapping and adaptors in the outer clathrin coat. membrane receptors. arrangement © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior AP2 adaptors engage the cytoplasmic tails of membrane receptors and their cargo. Dynamin is a G protein required for the release of a clathrin-coated vesicle from the membrane where it forms. Dynamin promotes a GTP-mediated fission of the coated pit from the plasma membrane Dynamin acts as an enzyme followed by disassembly of the dynamin ring that uses the energy from GTP to provide mechanical force. ATPase Hsc70, recruited by EM: coated vesicle auxillin, is required to forming in presence of dissociate the clathrin coat. non-hydrolyzable GTP(S) © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Changes in protein conformation upon AP2 binding to the plasma membrane AP2 adaptors normally exist in the cytosol in a locked conformation. Binding of AP2 complex to PI(4,5)P2 causes a conformational change in AP2. The AP2 cargo binding site becomes exposed, allowing it to interact with specific membrane receptors. © 2013 John Wiley & Sons, Inc. All rights reserved.  The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior LDL receptor EGF receptor The Endocytic Pathway – After internalization, vesicle-bound materials are transported in vesicles and tubules known as endosomes. – Early endosomes are EM: internal vesicles in the located near the lumen of a late endosome periphery of the cell. It sorts materials and sends bound ligands to the late endosomes. – Late endosomes are near the nucleus, also known as multivesicular bodies (MVBs). Endocytosis of receptor– EM: Gold particles bound to internalized EGF receptors ligand complexes © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior LDLs and Cholesterol Metabolism – Low-density lipoproteins (LDLs) are a complex of cholesterol and proteins. – LDL receptors are transported to the plasma membrane and bound to a coated pit. Each particle consists of approximately 1500 – LDLs are taken up by cholesterol ester molecules surrounded by a RME and taken to the monolayer of P-lipids and cholesterol and a lysosomes, releasing the single molecule of the protein apolipoprotein B cholesterol for use by which interacts specifically with LDL receptor the cells. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior A model for atherosclerotic plaque formation LDLs and cholesterol metabolism – High-density lipoproteins (HDLs) transport cholesterol from tissues to the liver for excretion. – HDLs associated with lowering cholesterol levels; LDLs associated with high blood cholesterol. – LDL deposition leads to plaques on inner lining of blood vessels. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Phagocytosis Phagocytosis: The process of engulfment as illustrated by a polymorphonuclear leukocyte ingesting a yeast cell (lower left). Phagocytosis – For the uptake of large particles. – The plasma membrane takes up a particle and pinches off to form a phagosome. – The phagosome fuses with a lysosome and the material is digested within the phagolysosome. © 2013 John Wiley & Sons, Inc. All rights reserved. The Endocytic Pathway: Moving Membrane and Materials Into the Cell Interior Phagocytosis The engulfment of particles by phagocytosis is driven by actin- containing microfilaments. Not all bacteria engulfed by phagocytic cells are destroyed; some species hijack the phagocytic machinery for their own survival. – Mycobacterium tuberculosis (affects fusion with lysosome) – Coxiella burnetti (pH tolerant so can survive in the lysosome) – Listeria monocytogenes (can degrade the lysosome) Summary of the phagocytic pathway © 2013 John Wiley & Sons, Inc. All rights reserved.  Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts Proteins targeted to nuclei, mitochondria, chloroplasts and peroxisomes contain signal sequences that serve as addresses. Uptake of Proteins into Peroxisomes is mediated by a peroxisomal targeting signal (PTS). Uptake of Proteins into the Mitochondria: – Most proteins contain a targeting sequence called pre-sequence. © 2013 John Wiley & Sons, Inc. All rights reserved.  Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts Uptake of Proteins into Mitochondria – The protein must be presented unfolded. – The outer mitochondrial membrane includes a protein- import complex (TOM complex) which includes a receptor and channel. – Proteins destined for the inner mitochondrial membrane engage with another protein- import complex (TIM complex), which includes two major complexes: TIM22 and TIM23. Importing proteins into a mitochondrion © 2013 John Wiley & Sons, Inc. All rights reserved.  Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts 3D-model of the Uptake of Proteins mitochondrial protein- into Mitochondria import machinery, – Movement into showing the number, the matrix is relative size, and voltage- topology of the dependent. various proteins involved in this – Chaperones are activity. involved in unfolding the TOM complex (red), protein and later TIM23 complex refolding it inside (yellow-green), TIM22 the complex (green), and mitochondrion. chaperones (blue). © 2013 John Wiley & Sons, Inc. All rights reserved.  Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts Uptake of Proteins into Chloroplasts – Most chloroplast proteins are imported form the cytosol. – Outer and inner envelope membranes contain translocation complexes (Toc and Tic) to facilitate import of the proteins. – Chaperones unfold proteins in cytosol and fold them in the chloroplasts. – Proteins include a transit peptide sequence. Importing proteins into a chloroplast © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis A handmade A high-power model of an electron “empty basket” micrograph of that would form an empty the surface proteinaceous lattice of a basket. coated vesicle. The mechanism of endocytosis was first proposed in the 1960s to explain the uptake of yolk proteins during oocyte growth. Insights into the structure of coated vesicles were revealed using electron microscopy. © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis HMG CoA reductase activity in normal fibroblasts was measured following addition of the lipoprotein fraction of calf serum (open squares), unfractionated calf serum (closed circles), or nonlipoprotein fraction of calf serum (open triangles). In 1970s clathrin was identified as the predominant protein in coated vesicles. Cells from individuals with familial hypercholesterolemia (FH) are unable to regulate cholesterol biosynthesis in response to LDL. Brown and Goldstein demonstrated that those affected with FH had a defect in RME of LDL. © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis Fibroblast cells from either a control subject (closed circles) or a patient with homozygous FH (open circles) were grown in dishes containing fetal calf serum. At the indicated time, extracts were prepared, and HMG CoA reductase activity was measured. In 1970s clathrin was identified as the predominant protein in coated vesicles. Cells from individuals with familial hypercholesterolemia (FH) are unable to regulate cholesterol biosynthesis in response to LDL. Brown and Goldstein demonstrated that those affected with FH had a defect in RME of LDL. © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis Time course of radioactive [125I]-labeled LDL binding to cells from a normal subject (circles) and a homozygote with FH (triangles) at 37oC. In 1970s clathrin was identified as the predominant protein in coated vesicles. Cells from individuals with familial hypercholesterolemia (FH) are unable to regulate cholesterol biosynthesis in response to LDL. Brown and Goldstein demonstrated that those affected with FH had a defect in RME of LDL. © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis Electron micrographs showed that cells of individuals with FH encode a receptor unable to bind LDL. Analysis of the mutant receptor showed a single amino acid substitution. Electron micrograph showing the binding of LDL to coated pits of human fibroblasts. © 2013 John Wiley & Sons, Inc. All rights reserved.  Experimental Pathways: Receptor-Mediated Endocytosis A series of fluorescence images showing the capture of a single red-fluorescent LDL particle by a green-fluorescent clathrin-coated pit and its incorporation into a clathrin- coated vesicle, which becomes uncoated and moves into the cytoplasm. © 2013 John Wiley & Sons, Inc. All rights reserved.  Synopsis The cytoplasm of eukaryotic cells contains a system of membranous organelles, including the endoplasmic reticulum, Golgi complex, and lysosomes, that are functionally and structurally related to one another and to the plasma membrane. The endoplasmic reticulum (ER) is a system of tubules, cisternae, and vesicles that divides the cytoplasm into a luminal space within the ER membranes and a cytosolic space outside the membranes. Proteins to be synthesized on membrane-bound ribosomes of the RER are recognized by a hydrophobic signal sequence, which is usually situated near the N- terminus of the nascent polypeptide. Most of the lipids of a cell’s membranes are also synthesized in the ER and moved from that site to various destinations. The addition of sugars (glycosylation) to the asparagine residues of proteins begins in the rough ER and continues in the Golgi complex. © 2013 John Wiley & Sons, Inc. All rights reserved.  Synopsis The Golgi complex functions as a processing plant, modifying the membrane components and cargo synthesized in the ER before it moves on to its target destination. The Golgi complex is also the site of synthesis of the complex polysaccharides that make up the matrix of the plant cell wall. Most, if not all, of the vesicles that transport material through the endomembrane system are encased initially in a protein coat. Several distinct types of coated vesicles have been identified. Each compartment along the biosynthetic or endocytic pathway has a characteristic protein composition. Vesicles that bud from a donor compartment possess specific proteins in their membrane that recognize proteins located in the target (acceptor) compartment. © 2013 John Wiley & Sons, Inc. All rights reserved.  Synopsis Lysosomes are diverse-appearing, membrane-bound organelles that contain an array of acid hydrolases capable of digesting virtually every type of biological macromolecule. The vacuoles of plants carry out a diverse array of activities. Endocytosis facilitates the uptake of fluid and suspended macromolecules, the internalization of membrane receptors and their bound ligands, and functions in the recycling of membrane between the cell surface and cytoplasm. Phagocytosis is the uptake of particulate matter. Proteins within peroxisomes, mitochondria, or chloroplasts that are encoded by nuclear genes must be imported into the organelle posttranslationally. © 2013 John Wiley & Sons, Inc. All rights reserved. Copyright 2013 John Wiley & Sons, Inc. All rights reserved. Reproduction or translation of this work beyond that permitted in section 117 of the 1976 United States Copyright Act without express permission of the copyright owner is unlawful. Request for further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. The purchaser may make back-up copies for his/her own use only and not for distribution or resale. The Publisher assumes no responsibility for errors, omissions, or damages caused by the use of these programs or from the use of the information herein. © 2013 John Wiley & Sons, Inc. All rights reserved.