05_Cellular organelles_2024.pdf

Cellular Organelles & Functions: Intracellular Organelles Lecture Notes 5 Gerald Karp, Chapter 8 Alberts, Chapter 12, 13 1 Specific Aims: l Specific Aim #1: Understand overall intracellular st...

Cellular Organelles & Functions: Intracellular Organelles Lecture Notes 5 Gerald Karp, Chapter 8 Alberts, Chapter 12, 13 1 Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 2  A cell consists of multiple intracellular components/ organelles l Mitochondria (power plant) l Endoplasmic reticulum l Golgi complex Endo- membrane l Endosomes system l Lysosomes l Each compartment is surrounded by membrane l Inside membrane, environment is very different from cytosol l Each compartment has unique functions 3 Eukaryotic cells Transmission electron microscopy (TEM) image 4 Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 5 Mitochondria is the energy generator in cells l Center of oxidative metabolism: l Generates energy needed for cellular activities and stored as ATP l Incorporates O2 into metabolism to oxidize cellular compounds into CO2 and water l Dynamic structure that can change shape and fuse together or split to 2 l Occupy 15-20 % cell volume in liver, contain more than a thousand proteins 6  Membranes divide mitochondria into 2 aqueous compartments l Matrix: gel-like containing many enzymes, ribosomes & mitochondrial DNA l Þ mitochondria possess own genetic material & machinery to make its own RNA & proteins l Human mitochondria DNA encodes 2 ribosomal RNA and 22 tRNAs used in protein synthesis within organelle l Intermembrane space (between outer and inner membrane): contains substances similar to cytoplasm, and is the location for energy reactions l Cristae contain large membrane surface that houses machinery needed for 7 aerobic respiration and ATP formation Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 8 Endomembrane system makes up the network in which materials are transported within cell l Matters are transported in vesicles that are membrane bound. l Movement are directed and moved by motor proteins operating on cytoskeleton. l 2 main pathways: l Endocytic pathway (into cell) l Matters uptaken by cell move to compartments e.g. endosome, lysosome 9  Endomembrane system makes up network in which materials are transported within cell l Biosynthetic pathway/ secretory pathway (out of cell) l Proteins made in ER, modified during passage through Golgi complex, transported from Golgi to other parts of cell, e.g. plasma membrane. l Majority discharged from cell, \ secretory pathway l A) constitutive secretion: discharge to ECM continuously l B) regulated secretion: response to stimulus e.g. neurotransmitters stored in larger membrane-bound secretory 10 granules Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 11 Endoplasmic reticulum (ER) l Rough ER: l Ribosomes attached Þ protein synthesis l Site of biosynthesis l Smooth ER: l Lacks ribosomes l Synthesis of hormone (lipids) l Release of glucose from glycogen l Sequestering Ca2+ 12 Rough ER l Ribosomes attached to cytosolic surface Þ protein synthesis l Composed of flattened sacs (cisternae) l Continuous with outer membrane of nuclear envelope l Function: starting point of biosynthetic pathway, site of synthesis of proteins, carbohydrate chains, phospholipids 13 Rough ER is the site for synthesis of polypeptides that are usually: l Secreted proteins l Integral membrane proteins l Soluble proteins that reside in compartments of endomembrane system, e.g. ER, Golgi complex lysosomes, endosomes, vesicles l In contrast, there are also ‘free’ ribosomes (not attached to RER) where proteins are made too (mainly proteins that remain in cells) l Site of protein synthesis is determined by amino acid sequence in N-terminal of polypeptide l Þ secretory proteins contain signal sequence that directs them to RER ribosomes 14 Rough ER is a major protein processing plant l Synthesis of secretory, lysosomal proteins (mostly glycoproteins) on membrane bound ribosomes Signal recognition particle l ER packed with molecular chaperones – control protein folding l Allows processing of newly synthesized proteins to be folded 15 properly, modified and dispatched to target destination 16 Synthesis of secretory, lysosomal proteins on membrane bound ribosomes 1. mRNA binds to free ribosome ® protein synthesis starts 2. Signal sequence (6-15) of hydrophobic AA emerges. This acts as signal that directs polypeptide to ER membrane 3. Signal sequence recognized by signal recognition particle (SRP). SRP binds signal sequence and ribosome stopping further synthesis 4. Complex bind specifically to SRP receptor on ER 5. Protein-lined channel embedded in ER membrane, translocon, binds to ribosome 6. SRP detaches, protein synthesis continues through translocon into ER lumen. Plug proposed to seal channel to prevent unwanted passage of Ca2+ and other ions 7. When translocation completed, ribosome released for reuse, and plug reinserted 17 Role of rough ER – synthesis of integral membrane proteins l Same initial as in secretory protein. l But unlike soluble secretory & lysosomal proteins that passes through entirely the ER membrane during translocation, integral proteins contain hydrophobic transmembrane segments that are shunted directly from channel of translocon into lipid bilayer 18 Protein synthesis - ER 19 Glycosylation in RER Sugars are added up to the required structure and the whole structure is then added onto protein 20 Glycosylation in RER l Lipid carrier, dilichol phosphate, is where sugars are added one by one l In mammalian cells process of attaching core oligosaccharide is invariant: 1. Start with transfer of N-acetyl-glucosamine 1-phosphate 2. Transfer of another N-acetyl-glucosamine 1-phosphate 3. Transfer of 9 mannose 4. Transfer of 3 glucose 5. Transfer bulk oligosaccharide to nascent polypeptide Þ ER makes only 1 type of core oligosaccharide: N-linked oligosaccharide (O-linked made in Golgi complex) Þ core / bulk oligosaccharide can be modified later in Golgi complex to give unique oligosaccharide chain 21 Synthesis of most membrane lipids takes place entirely in ER and is later modified l Membrane grow as newly synthesized proteins and lipids are fused with existing membrane in ER l Membrane components move from ER to other compartments in cell l Þ lipids modified as they move from ER to other parts of cells l Phospholipid layers of membrane are asymmetrical starting from ER. l Þ components in cytosol always remain in cytosol. If faces lumen, always faces lumen l Þ lumen environment is similar to extracellular space 22 Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 23 Golgi Complex modifies proteins from ER l Protein from ER next moves to Golgi complex l Cis-most face consists of interconnected network of tubules – cis-Golgi network (CGN) – function as sorting station to distinguish between proteins to go back to ER or to advance into Golgi Cis: face complex closest to ER Trans: exit l Trans-Golgi network (TGN) face of Golgi also sorts proteins to different complex types of vesicles 24 Glycosylation in Golgi complex l Glycoproteins may be modified as they pass through Golgi (most mannose removed, other sugars added) l Sequence of sugars added depends on spatial arrangement of specific glycotransferases that contacts the glycoproteins as it passes through Golgi stack l Golgi complex makes most of cell’s complex oligosaccharides * main difference is that glycosylation is varied, unlike ER where only 1 core 25 oligosaccharide is assembled * Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 26  Types of vesicle transport & their function l Transport vesicles are covered with protein coat to allow selection of cargo l Three types of protein coats: l 1) COPII (coat proteins) coated vesicles: move materials forward from ER to Golgi l 2) COPI-coated vesicles: move materials backward from Golgi to ER, from trans Golgi cisternae to cis Golgi cisternae l 3) Clathrin-coated vesicles: move materials from TGN to endosomes, lysosomes, move materials from plasma membrane to cytoplasmic compartments, move from endosomes to lysosomes 27 ERGIC: ER Golgi Intermediate Compartment Retaining & retrieving resident ER proteins l Protein level maintained because: 1. not all included in transport vesicles 2. ‘escaped’ molecules retrieved back because AA sequence on protein structure that acts as retrieval signal: ‘Lys-asp-glu-leu’ (KDEL) (lumen ER proteins) lysine-aspartic acid-glutamic acid-leucine l KDEL receptor recognizes KDEL on ER resident proteins. Binds that protein and transport it back to ER from Golgi in COPI-coated vesicles 28 Cellular organelles 29 Endomembrane system 30 Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 31 Endocytic pathway describes the movement of membrane and materials INTO cell l 2 basic processes of endocytic pathway: l Endocytosis: cell internalizes cell surface receptors and bound extracellular (EC) ligands l Phagocytosis: uptake of particulate matter l Endocytosis has 2 main categories: l Pinocytosis (bulk phase endocytosis): non-specific uptake of extracellular fluids. Any molecules near also uptaken. (may function as plasma membrane recycling mechanism mainly) l Receptor-mediated endocytosis: specific uptake after binding of ligands to their receptors 32 Receptor-mediated endocytosis (RME) and role of coated pits l Selective efficient uptake. Low concentration molecules can be uptaken l Ligands bound to receptors that collect in specialized domains of plasma membrane called coated pits l Receptor concentrated at 10-20 times l Looked ‘pitty’ under EM: because receptors covered by clathrin protein 33  Endocytic pathway l Endocytosis of housekeeping receptor (binds to materials that will be used by cells constantly) – typically leads to delivery of bound materials into cell and return of receptor to cell surface for 2nd round l Endocytosis of signaling receptors (binds to hormones or growth factors) – leads to destruction of receptor after molecule transport, therefore decrease in cellular response to signaling molecules l Internalized material delivered to 34 endosomes Receptor mediated endocytosis 35 Endocytic pathway l Steps: 1. Ligands bind to receptors 2. Receptors internalized are transported in vesicles to early endosomes 3. Ligand from HK receptor released 4. HK receptor recycled 5. Ligands from HK receptor and signaling receptor in sorting compartment of early endosome are dispatched to late endosome 6. Then to lysosome Housekeeping (HK) receptor: binds to biomolecules constantly required by cells, 36 e.g. low-density lipoprotein (LDL, cholesterol) Endosomes act as distribution centers l 2 distinct classes: l Early endosomes – located near peripheral region of cell l Late endosomes – located near nucleus l Difference lies in pH, protein composition, buoyant density l Early endosome act as sorting station. HK receptors are dissociated from ligands by acidic endosomal environment and recycled l Released ligands concentrated in sorting compartment and dispatched to late endosome 37 Phagocytosis involves large particles l Cell eating by few cell types (macrophages/ neutrophils) l Large particles > 0.5µm diameter l Defense mechanism against invading organism, damaged / dying cells and debris l Microorganism in cells killed by lysosomal enzyme or O2 free radicals in 38 lumen of phagosome Phagocytic pathway 1. Enclose organism / particle within folds of plasma membrane 2. Folds fuse to produce phagosome (vacuole) pinches off into cell 3. Phagosome fuses with lysosome (phagolysosome) and cargo digested Bacteria mechanism against body’s defense: 1. stops vacuoles from fusing with lysosome 2. enter lysosome but resistant to enzyme degradation 39 3. destroys lysosomal membrane 40 Specific Aims: l Specific Aim #1: Understand overall intracellular structure l Specific Aim #2: Understand the structure & function of mitochondria l Specific Aim #3: Understand overall structure of endomembrane system l Specific Aim #4: Understand the components of endomembrane system, structure and function – ER l Specific Aim #5: Understand the components of endomembrane system, structure and function – Golgi complex l Specific Aim #6: Understand the components of endomembrane system, structure and function – Vesicles / vesicle transport l Specific Aim #7: Understand the components of endomembrane system, structure and function – endosomes l Specific Aim #8: Understand the components of endomembrane system, structure and function - lysosomes 41 Lysosomes’ major role involves breaking down materials brought into cell from ECM l Also breaks down cell organelle (autophagy). Organelle to be broken down is surrounded by a double membrane derived from an ER cisternae. Outer membrane fuse with lysosome (autophagolysosome) Þ enclosed organelle broken down l Þ cell energy source (one form) l Þ removal of damaged proteins and organelles l Þ residual body exocytosed or kept in cells as lipofuscin granules (amount in cells reflect 42 age of cells)  Lysosomes are animal cells’ digestive organelles l Typically has at least 50 different hydrolytic enzymes produced in RER and targeted to lysosomes. l Low pH in lysosomes ~4.6 (maintained by proton pumps) l Membranes have highly glycosylated integral proteins whose carbohydrate chains thought to protect membrane from enzymes inside lumen 43 Left: autophagosome with mitochondria & peroxisome; right: lysosomes Specific Aims - Summary l Specific Aim #1: Understand that cellular organelles include mitochondria, nucleus, ER, endosomes, lysosomes, Golgi complex, etc. l Specific Aim #2: Understand that mitochondria is the cell’s power plant l Specific Aim #3: Understand that endomembrane system comprises of endocytic pathways & secretory pathways l Specific Aim #4: Understand that ER is the site for polypeptides (secretory, lysosomal and integral membrane proteins & membrane lipids) synthesis l Specific Aim #5: Understand that protein glycosylation and dispatch occur in Golgi complex l Specific Aim #6: Understand that biomolecules are transported in vesicles within, into and out of cells l Specific Aim #7: Understand that endosomes act as distribution centers for molecules uptaken by cells l Specific Aim #8: Understand that lysosomes are animal cells’ digestive organelles 44