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Chapter 12: Cellular Organelles and Membrane Trafficking

• This chapter emphasizes the dynamic nature of the endomembrane system within eukaryotic cells.
• It distinguishes between regulated and constitutive secretion.
• It details the structure and function of the rough and smooth endoplasmic reticulum (ER).
• Events in synthesis and transport of membranes and proteins throughout the cell are outlined, including glycosylation processing of secretory/integral membrane proteins.
• The structure, function, and polarization of the Golgi complex are examined.
• The role of coated and non-coated vesicles in membrane trafficking is described.
• The signals used to target proteins to their cellular location are explained.
• The steps involved in exocytosis and its triggers are detailed.
• Lysosomal structure, function, and diseases caused by lysosome malfunction are discussed.
• Distinctions between phagocytosis, bulk phase endocytosis, and receptor-mediated endocytosis are made.

Introduction

• Membranes compartmentalize the cytoplasm of eukaryotic cells.
• The endomembrane system (ER, Golgi complex, endosomes, lysosomes, and vacuoles) functions as a coordinated unit.

Overview of the Endomembrane System

• Organelles of the endomembrane system are part of an integrated network, shuttling materials back and forth.
• Materials are shuttled between organelles via membrane-bound transport vesicles.
• Upon reaching their destination, vesicles fuse with the acceptor compartment's membrane.

Overview of the Endomembrane System: Biosynthetic and Secretory Pathways

• Several distinct pathways for material transport through the cytoplasm have been defined.
• Biosynthetic pathway: synthesis, modification, and transport of proteins within the cell.
• Secretory pathway: when proteins are discharged (secreted) from the cell.
• Constitutive secretion: continuous process.
• Regulated secretion: in response to a stimulus.
• Endocytic pathways unite endomembranes into a dynamic, interconnected network.

Overview of Endomembrane System: Synthesis and Transport of Secretory Proteins

• Materials to be secreted are stored in large, membrane-bound secretory granules.
• An example of this is the secretion of digestive enzymes by pancreatic cells.

Study of Cytomembranes: Autoradiography

• Autoradiography is a method to visualize biochemical processes using radiolabeled materials exposed to a photographic film.
• This technique, now largely superseded by fluorescent-based approaches, can determine the locations where secretory proteins are synthesized within cells.

Study of Cytomembranes: GFP-Based Protein Tracking

• The use of green fluorescent protein (GFP) reveals protein movement within living cells.
• GFP-DNA chimeras allow observation of protein synthesis in cells.
• Fusing viral genes to GFP allows for the large-scale production of proteins, which is useful for studying protein traffic.

Study of Cytomembranes: Biochemical Analysis of Subcellular Fractions

• Techniques to homogenize cells and isolate organelles can be separated through sub-cellular fractionation.
• Techniques can further characterize membrane vesicles derived from the endomembrane system, forming microsomes for further study.

Study of Cytomembranes: Use of Cell-Free Systems

• Cell-free systems, lacking whole cells, provide information about the roles of proteins in membrane trafficking.

Study of Cytomembranes: Utility of Genetic Mutants

• Genetic mutants provide insights into the functions of normal gene products.
• Isolation of proteins from yeast has led to the identification of homologous proteins in mammals, demonstrating the conserved nature of endomembrane systems.

Study of Cytomembranes: Utility of Genetic Mutants: Studying Mutants Using RNA Interference

• RNA interference (RNAi) is a process in which cells produce small RNAs that bind to specific mRNAs, inhibiting their translation into proteins.
• Scientists can determine involved genes in specific processes by identifying which siRNAs interfere with the process.

The Endoplasmic Reticulum

• The endoplasmic reticulum (ER) is a network of membranes that permeates much of the cytoplasm.
• Like other organelles, the ER is highly dynamic and continually undergoes turnover and reorganization.
• The ER is divided into two subcompartments: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER).
• The composition of the luminal space of the ER membranes differs from the surrounding cytosol.

The Endoplasmic Reticulum: Functions of the RER

• Composed of a network of flattened sacs (cisternae).
• Continuous with the outer membrane of the nuclear envelope and contains ribosomes on its cytosolic surface.
• Various cell types have different ratios of RER to SER depending on cell activity.

The Endoplasmic Reticulum: Functions of the SER

• Extensively developed in a number of cell types, with functions including steroid hormone synthesis in endocrine cells, detoxification in the liver of various organic compounds (P450 enzymes), and sequestration of calcium ions in muscle cells.

The Endoplasmic Reticulum: Functions of the RER: Synthesis of Proteins on Membrane-Bound versus Free Ribosomes

• Approximately one-third of the polypeptides encoded by the human genome are synthesized on RER ribosomes.
• These include secreted proteins, integral membrane proteins, and soluble proteins within organelles.
• Polypeptides synthesized on free ribosomes include cytosolic proteins, peripheral membrane proteins, nuclear proteins, and proteins incorporated into chloroplasts, mitochondria, and peroxisomes.

The Endoplasmic Reticulum: Site of Synthesis

• A signal sequence at the N-terminus of a protein determines its synthesis site.
• The signal sequence attaches to the secretory protein. Proteins travel into the ER cisternal space via a protein-lined membrane pore, either co-translationally or post-translationally.

The Endoplasmic Reticulum: Synthesis of Proteins on Membrane-Bound Ribosomes

• Messenger RNA (mRNA) binds to free ribosomes in the cytosol.
• Secretory proteins synthesized on membrane-bound ribosomes have their signal sequences recognised by a signal recognition particle (SRP).

The Endoplasmic Reticulum: Binding to the ER

• Binding to the ER occurs through two sequential interactions: Initially, the SRP interacts with the SRP receptor; then, the ribosome-nascent peptide chain complex binds to the ER via the translocon.
• Release of SRP requires guanosine triphosphate (GTP)-binding proteins (G proteins).

The Endoplasmic Reticulum: 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 oligosaccharyltransferases.
• Chaperones in the RER lumen assist in protein folding and disulfide bond formation in cysteine residues.

The Endoplasmic Reticulum: Synthesis of Integral Membrane Proteins

• Integral membrane proteins contain hydrophobic transmembrane segments.
• The translocon assists in the proper orientation of transmembrane sequences.
• The orientation of the first transmembrane segment determines the arrangement of the protein within the membrane.

The Endoplasmic Reticulum: Membrane Biosynthesis in the ER

• Membranes arise from pre-existing membranes.
• Lipids are inserted into existing membranes.
• As membranes move between compartments, proteins and lipids are modified.
• Membrane asymmetry is initially established and maintained during trafficking.

The Endoplasmic Reticulum: Modifying the Lipid Composition of Membranes

• Most membrane lipids are synthesized in the ER, except for sphingomyelin and glycolipids found in mitochondria and chloroplasts.
• Newly synthesized phospholipids are inserted into one half of the bilayer and then flipped to the opposite leaflet by flippases.
• Enzymes modify pre-existing lipids within the membrane.

The Endoplasmic Reticulum: Modifying lipid Composition of Membranes: Contributing Factors to Organelle Lipid Contribution

• Organelle-specific enzymes catalyze lipid conversion.
• Inclusion and exclusion processes occur during vesicle formation.
• Lipid-transfer proteins transport lipids without vesicle transport.

The Endoplasmic Reticulum: Glycosylation in the RER

• The addition of sugars (glycosylation) is facilitated by glycosyltransferases.
• Core carbohydrate segments are assembled on lipid carriers (dolichol phosphate) and subsequently transferred to the polypeptide.
• Oligosaccharyltransferase modifies core carbohydrates.

The Endoplasmic Reticulum: Glycosylation, Quality Control

• A glycoprotein undergoes quality control to ensure its fitness for its specific compartment.
• Misfolded proteins are tagged with a terminal glucose and recognized by chaperones for refolding.
• If a protein does not correctly fold, it is sent to the cytosol and destroyed.

The Endoplasmic Reticulum: Mechanisms for Destruction of Misfolded Proteins

• Misfolded proteins are not destroyed in the ER, but rather transferred to the cytosol and degraded within proteasomes (ER-associated degradation). This process(ERAD) prevents misfolded proteins from reaching the cell surface.

The Endoplasmic Reticulum: Destruction of Misfolded Proteins: The Unfolded Protein Response (UPR)

• The accumulation of misfolded proteins triggers the unfolded protein response (UPR).
• Sensors in the ER are kept inactive by chaperone BiP.
• If misfolded proteins accumulate, BiP molecules become unable to inhibit the sensors.
• Activated sensors trigger proteins responsible for the destruction of misfolded proteins.

The Endoplasmic Reticulum: Visualizing Membrane Traffic with the Use of a Fluorescent Tag

• Movement of vesicular-tubular carriers from the ER to the Golgi, using VSV-G:GFP tags.
• RER have specialized exit sites for the formation of transport vesicles (no ribosomes).
• Transport vesicles fuse to form the ERGIC (endoplasmic reticulum Golgi intermediate compartment), which moves toward the Golgi complex.

The Golgi Complex

• The Golgi complex consists of a stack of flattened cisternae.
• It is divided into several functionally distinct compartments.
• The cis face of the Golgi is positioned towards the ER, and the trans face is on the opposite side of the stack.

The Golgi Complex: Glycosylation of proteins in the Golgi Complex

• Assembly of carbohydrates within glycolipids and glycoproteins takes place in the Golgi.
• The order of sugar incorporation is determined by glycosyltransferases.
• Glycosylation steps can be diverse.

The Golgi Complex: Movement of Materials through the Golgi Complex

• The vesicular transport model describes cargo as shuttled between CGN and TGN in vesicles.
• In the cisternal maturation model, cisternae mature as they move from the cis face to the trans face.
• The current model is similar to the cisternal maturation model but includes vesicle retrograde transport.

Types of Vesicle Transport and Their Functions: Coated Vesicles

• Materials are carried between compartments using coated vesicles.
• Protein coats have dual functions:
• -Curve the membrane to form a vesicle.
• -Select components to be transported.

Types of Vesicle Transport and Their Functions: COPII-Coated Vesicles

• Move materials from the ER to the ERGIC and Golgi complex.

Types of Vesicle Transport and Their Functions: COPI-Coated Vesicles

• Move materials from the ERGIC or Golgi to the ER, or from the trans Golgi to the cis Golgi cisternae.

Types of Vesicle Transport and Their Functions: Clathrin-Coated Vesicles

• Move materials from the TGN to endosomes, lysosomes, and plant vacuoles.

Types of Vesicle Transport and Their Functions: COPII-Coated Vesicles: Transporting Cargo from the ER to the Golgi Complex

• Select proteins involved in enzyme and membrane function.
• A small G protein (Sar1) plays a regulatory role in vesicle assembly.
• Sar1 binds to the ER; disassembly is triggered by GTP hydrolysis, producing Sar1-GDP.

Types of Vesicle Transport and Their Functions: COPI-Coated Vesicles: Transporting Escaped Proteins Back to the ER

• Adenosylation ribose factor 1 (Arf1) is a G protein required for vesicle transfer between cisternae.
• Protein retention in organelles is achieved via two mechanisms: -Retention of resident molecules excluded from transport vesicles. -Retrieval of "escaped" molecules back to their original compartment.

Types of Vesicle Transport and Their Functions: Resident Proteins of the ER

• These contain an amino acid sequence at the C-terminus that acts as a retrieval signal.
• Specific receptors capture molecules, bringing them into the ER within COPI-coated vesicles.

Types of Vesicle Transport and Their Functions: Beyond the Golgi Complex: Sorting Proteins at the TGN

• Lysosomal proteins are tagged with phosphorylated mannose residues.
• Tagged lysosomal enzymes are recognized and captured by mannose 6-phosphate receptors (MPRs).

Types of Vesicle Transport and Their Functions: Sorting and Transport of Non-Lysosomal Proteins

• Secretory proteins aggregate in dense granules that emerge from 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.

Types of Vesicle Transport and Their Functions: Targeting Vesicles to a Particular Compartment

• Rab proteins (G proteins) regulate vesicle docking.
• SNAREs (v-SNAREs in vesicles and t-SNAREs in target membranes) mediate vesicle docking and fusion.
• Vesicle and target membranes fuse via interactions between t- and v-SNARE proteins.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Exocytosis

• Discharge of a secretory vesicle or granule after fusion with the plasma membrane.
• Triggered by an increase in [Ca2+].
• Vesicle and plasma membrane contact leads to "fusion pore" formation.
• The luminal vesicle membrane becomes the outer PM surface, and the cytosolic vesicle membrane becomes part of the inner PM surface.

Lysosomes

• Lysosomes contain acid hydrolases for digesting all types of biological molecules.
• Low pH optimum is maintained by a proton pump (H+-ATPase).

Lysosomes: Autophagy and Human Perspective: Lysosomal Storage Disorders

• Autophagy: Organelles are surrounded by a double membrane (autophagosome) and then fused with a lysosome (autophagolysosome) to digest and recycle the component.
• Lysosomal storage disorders result from the absence of specific enzymes, causing undigested materials to accumulate. This can give rise to a wide variety of symptoms.
• Tay-Sachs disease, a well-studied disorder, results from a deficiency in an enzyme responsible for degrading gangliosides - major cell membrane components.

The Endoplasmic Reticulum: Plant Cell Vacuoles

• Vacuoles are membrane-bound, fluid-filled compartments that serve diverse storage functions.
• Plant vacuoles contain active transport systems to maintain a high ion concentration within the vacuole and allows water to enter by osmosis.
• Plant vacuoles also contain acid hydrolases.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Endocytosis, Pinocytosis, Phagocytosis

• Endocytosis is the uptake by cells of ligands and extracellular fluids.
• Pinocytosis is the non-specific uptake of extracellular fluids.
• Phagocytosis is the uptake of particulate matter.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Receptor-Mediated Endocytosis

• Substances entering the cell through coated pits on the plasma membrane are bound to coated pits.
• Clathrin-coated regions invaginate into the cytoplasm, forming vesicles that detach and become free from the cytoplasm.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Receptor Recognition

• Receptor-mediated endocytosis (RME) involves receptors capturing extracellular ligands, and moving these to the cell interior through coated vesicles.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Geometric Feature

• Coated pits viewed from the cytoplasm are polygon-shaped, resembling a honeycomb structure.
• The honeycomb design of the structure arises from the arrangement of clathrin blocks.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Receptor-Mediated Endocytosis

• Clathrin contains three light and three heavy chains that form a triskelion.
• Coated vesicles contain adaptor proteins between clathrin and the membrane.
• AP2 is the most common clathrin adaptor.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Dynamin and Receptor-Mediated Endocytosis

• Dynamin is a G protein needed for releasing clathrin-coated vesicles from membranes.
• Dynamin acts as an enzyme, using GTP energy for mechanical work.
• Auxiliary ATPase Hsc70, with the aid of auxillin, is needed for disassembling the clathrin coat after vesicle formation.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: AP2 Adaptors and Receptors

• AP2 adaptors exist within the cytosol, in a locked conformation.
• AP2 complex binding to PI(4,5)P₂ triggers a conformational change in AP2.
• The AP2 cargo-binding site is exposed, enabling interaction with specific membrane receptors.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: The Endocytic Pathway

• After internalization, vesicle-bound materials are transported through vesicles and tubules (endosomes).
• Early endosomes are near the cell periphery, sorting materials and sending bound ligands to the late endosomes.
• Late endosomes are located near the nucleus, also known as multivesicular bodies (MVBs).

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: LDL and Cholesterol Metabolism

• Low-density lipoproteins (LDLs) are cholesterol and protein complexes.
• LDL receptors are transported to the plasma membrane and bind to coated pits.
• LDLs are taken up by receptor-mediated endocytosis (RME) and transported to lysosomes; cholesterol is released for cellular use.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior:LDL and Cholesterol Metabolism: A Model for Atherosclerotic Plaque Formation

• High-density lipoproteins (HDLs) transport cholesterol from tissues to the liver for excretion.
• HDLs contribute to lowering cholesterol levels, while LDLs are associated with high blood cholesterol and LDL deposition within blood vessel walls.

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior: Phagocytosis

• Phagocytosis is the process of particle ingestion by cells.
• Phagocytosis is driven by actin-containing microfilaments.
• Some bacteria hijack phagocytic machinery for survival (e.g., M. tuberculosis, C. burnetii, L. monocytogenes).

Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts: Uptake of Proteins into Mitochondria

• Proteins destined for mitochondria must be unfolded.
• The outer mitochondrial membrane contains TOM complex (receptor and channel), which facilitates protein import.
• Proteins destined for the inner membrane engage with TIM complex (TIM22 and TIM23), which facilitates import into the inner membrane.

Posttranslational Uptake of Proteins by Peroxisomes, Mitochondria, and Chloroplasts: Uptake of Proteins into Chloroplasts

• Most chloroplast proteins are imported from the cytosol.
• Outer and inner envelope membranes contain translocation complexes (Toc and Tic), facilitating protein import.
• Chaperones unfold proteins in the cytosol, folding them in chloroplasts.
• Proteins incorporate a transit peptide sequence for import.

Experimental pathways: Receptor-Mediated Endocytosis

• The mechanism of receptor-mediated endocytosis was proposed in the 1960s.
• Insights into the structure of coated vesicles were gained from electron microscopy.

Experimental pathways: Receptor-Mediated Endocytosis: Predominant Protein in Coated Vesicles

• Clathrin was identified as the predominant protein in coated vesicles and is involved in receptor-mediated endocytosis.

Experimental pathways: Receptor-Mediated Endocytosis in Familial Hypercholesterolemia (FH)

• Cells from individuals with familial hypercholesterolemia (FH) are unable to regulate cholesterol biosynthesis in response to LDL.
• Brown and Goldstein demonstrated that those with FH have a defect in LDL Receptor-Mediated Endocytosis (RME) of LDL.

Experimental pathways: Receptor-Mediated Endocytosis: Analysis of Mutant Receptor Structure

• Electron micrographs of FH individuals reveal defects in LDL receptor binding to coated pits, and analysis of mutant receptors indicates single amino acid substitutions as a cause of the disease.

Experimental pathways: Receptor-Mediated Endocytosis: Visualization of LDL uptake by fluorescence microscopy

• Fluorescence microscopy visualizing the capture of a single red-fluorescent LDL particle by green-fluorescent clathrin-coated pits.