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CELB30090 Advanced Cell Biology Prof. Jeremy C. Simpson Lecture 2 The endomembrane system Today’s lecture ... Overview of internal membranes Importance of subcellular compartmentalisation Introduction to the ER The Golgi complex The endosomal‐lysosomal system Peroxisomes The endomembrane system in cells The endomembrane system in cells Key points about subcellular membranes and their organisation The segregation of distinct biochemical reactions into separate membrane enclosed entities (organelles) results in a dramatic enhancement in efficiency A typical animal cell contains ca. 10 billion protein molecules that need to be co‐ordinated if the cell is to function correctly Allows conflicting reactions to be carried out in the same cell (eg: protein synthesis and protein degradation) Cells have evolved mechanisms that allow compartments to communicate with each other Proteins requiring transport between compartments must have appropriate signals that can be read by the cell Division into subcellular compartments allows pathways to be established (eg: secretory pathway) Internal protein transport pathways in cells Gated transport Via protein channels or pores eg: nuclear import and export Lecture 12 Transmembrane transport (translocation) Via protein translocation channels eg: protein entry into the ER Lecture 3 Vesicular transport Via membrane ‐bounded intermediates eg: transport from ER to Golgi Lectures 4‐7 The endomembrane system in cells The endomembrane system occupies a significant cell volume Relative volumes occupied by the major intracellular compartments in a liver cell (hepatocyte) The endomembrane system in cells Cell type and role determines relative abundance of organelles Relative amounts of membrane types in two kinds of eukaryotic cells The endomembrane system in cells Lipid composition is highly variable between organelles Possible evolution of the endomembrane system in eukaryotic cells Invagination of the plasma membrane Pinching off would result in a double membrane surrounding the ‘nucleus’ ‘ER’ would be continuous with the ‘nuclear envelope’ Engulfment of a bacterium Mitochondria have separate genome Mitochondria lumen remains isolated from rest of endomembrane system The endomembrane system in cells Endoplasmic reticulum (ER) (Lecture 3) Smooth ER – lipid synthesis, steroid hormone synthesis*, detoxification, storage of calcium ions Rough ER – protein synthesis, translocation, folding, glycosylation, antigen processing* Golgi complex / apparatus (Lecture 2) protein modification by glycosylation, completion of sphingomyelin and glycolipid synthesis Endosomes (Lecture 7) protein sorting between endo‐ and exocytic routes, sorting of receptors and ligands, signalling Lysosomes (Lectures 2 and 7) degradation processes, organelle turnover, antigen processing*, fertilisation* Peroxisomes (Microbodies) (Lecture 2) synthesis and degradation of hydrogen peroxide, oxidation of fatty acids, synthesis of plasmalogens*, photorespiration in plants * in specific cell types Co‐ordination of the endomembrane system using vesicular transport Secretory pathway / Biosynthetic pathway / Anterograde pathway / Exocytic pathway Endocytic pathway / Retrograde pathway The Golgi complex discovered by Camillo Golgi (Italian biologist, b. 1843) ‐ inventor of new types of staining procedures that he hoped might reveal the organization of nerve cells within the central nervous system applied a silver nitrate‐based stain for several days to cerebellum nerve cells and saw darkly staining reticular network near the cell nucleus (Nobel Prize in 1906) characteristic morphology ‐ flattened, disk‐like membranous cisternae with dilated rims and associated vesicles & tubules cisternae, typically 0.5 ‐ 1.0 µm diamater, are arranged in orderly stack like pancakes usually <8 cisternae are present per stack, but may have a few to several 1000 distinct stacks per cell, depends on cell type mammalian cell Golgi stacks are interconnected by membranous tubules to form a single, large ribbon‐like complex situated adjacent to the cell's nucleus 10 µm The Golgi complex Golgi cisternae polarized ‐ cis face (entry face closest to ER) ‐ trans face (exit face at opposite end of stack; closer to plasma membrane) new materials enter cis face and pass to trans face where they exit Golgi complex from the trans Golgi network (TGN) mechanism of transport through the Golgi is still strongly debated (Lectures 4 and 5) intrinsically linked to the cytoskeleton, motor proteins and matrix proteins The Golgi complex ‐ receives newly synthesised proteins from the ER, and processes them by proteolysis, amino acid modification (eg: hydroxylation), and by modifying their carbohydrate chains ‐ as glycoproteins pass though cis & medial Golgi cisternae, most of the mannose residues are removed from the core oligosaccharides ‐ other sugars are added sequentially by various glycosyltransferases (eg: N‐acetylglucosamine) ‐ sialyltransferase is only found in trans end of Golgi stack and adds sialic acid residues to the terminal of the chains ‐ the Golgi complex is wholly responsible for O‐linked glycosylation events Endosomal‐lysosomal system ‐ provides a link between the secretory and endocytic pathways ‐ major role in cargo sorting, recycling and degradation (Lecture 7) endosome carrier vesicle / multivesicular body) Gruenberg J and Stenmark H (2004) Nat Rev Mol Cell Biol 5:317‐323 Lysosomes ‐ typically contain at least 50 different hydrolytic enzymes made in RER and targeted for lysosomes ‐ can hydrolyze virtually every type of biological macromolecule, resulting in low MW products that can be transported across the lysosomal membrane into cytosol ‐ acidic interior (pH 4.6) maintained by proton ATPase pumps ‐ lysosomal morphology is neither distinctive nor uniform, they are dynamic organelles capable of rapid fusion and fission, often clustered in the peri‐nuclear area of the cell ‐ highly variable size range (25 nm to > 1 µm diameter) 10 µm Lysosomal functions ‐ general role in degradation of materials brought into cell ‐ specialised role in fertilization ‐ sperm head contains specialized lysosome (acrosome) ‐ organelle turnover (autophagy) Peroxisomes ‐ surrounded by only a single membrane, and do not contain DNA or ribosomes ‐ typical diameter of 0.1 – 1.0 µm ‐ present in all eukaryotic cells ‐ contain high concentrations of oxidative enzymes, such as catalase and urate oxidase, that are used to detoxify cells ‐ synthesis of plasmalogens – important phospholipid component of myelin sheaths 10µm Light microscope Electron microscope Peroxisomes ‐ contain high concentrations of oxidative enzymes, such as catalase and urate oxidase, that are used to detoxify cells ‐ oxidation reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various toxic molecules that enter the bloodstream ‐ ca. 25% of the ethanol we drink is oxidised to acetaldehyde in this way phenols formic acid formaldehyde alcohols Peroxisomes peroxisome biogenesis is not yet fully understood – likely a combination of being derived from the ER and self assembly peroxisome targeting signal: ‐Ser‐Lys‐Leu‐COO‐ (hydroxylated, positive, hydrophobic) at least 23 ‘peroxins’ so far identified participate in import defects in peroxin proteins (eg: Pex2) cause severe disease (eg: Zellweger syndrome) Key take home point Subcellular compartmentalisation allows eukaryotic cells to carry out more complex activities with a higher degree of efficiency