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CONCEPT: MEMBRANE ENCLOSED ORGANELLES Table of eukaryotic organelles and their functions Organelle Function % volume of cell Cytosol Aqueous fluid where metabolic pathways and chemical reactions occur 54 Nucleus Stores the genome; DNA/RNA synthesis 6 Endoplasmic Reticulum Synthesis of lipids and protein; Distribution of lipids and protein 12 Golgi Apparatus Modifications, sorting, and packaging of proteins/lipids 3 Lysosomes Intracellular degradation 1 Endosomes Sorting of incoming material 1 Mitochondria ATP synthesis by oxidative phosphorylation (powerhouse) 22 Chloroplasts (plants) ATP synthesis and carbon fixation by photosynthesis 22 Peroxisomes Oxidation of toxic molecules 1 Many organelles can be held in place, or throughout the cell using the cytoskeleton Motor proteins can move them through energy provided from ATP hydrolysis Creation of organelles occur through two main methods Endosymbiont theory describes the formation of mitochondria and chloroplasts All other organelles were created via invagination of the plasma membrane into small internal compartments - Endomembrane system organelles including Golgi, ER, peroxisomes, endosomes and lysosomes - Communication between these organelles occurs through small budding vesicles Page 2
EXAMPLE: Overview of the endomembrane system PRACTICE: 1. True or False: Organelles must remain stationary within the cell a. True b. False Page 3
2. Other than the cytoplasm, which organelle collectively makes up the largest volume of an animal cell? a. Nucleus b. Mitochondria c. Endoplasmic Reticulum d. Golgi Apparatus Page 4
CONCEPT: PROTEIN SORTING Proteins in cells are sorted through three mechanisms: gated transport, transmembrane transport, and vesicular transport Sorting signals (15-60 amino acids long) located on the direct its transport - Signal peptidases remove sorting signals after the protein arrives at the intended location - The protein remains in the cytosol if it lacks sorting signals 1. Gated Transport Gated transport refers to the transport of proteins and molecules between the cytosol and the nucleus The nuclear pore is responsible for this type of transport - Extends through the envelop - Internal pore structure is a meshwork of unstructured regions that prevent large molecules from diffusing Nuclear localization signals located on large molecules allow nuclear entrance or exit - Signal is recognized by import receptors which facilitate moving through the pore using energy from GTP EXAMPLE: Basic structure of a nuclear pore 2. Transmembrane Transport via Protein Translocators Protein translocators translocate proteins across membranes Translocates from the cytosol into the ER, mitochondria or chloroplasts Proteins must unfold in order to be translocated into their new location Page 5
- Signal sequences direct the protein to its location - Chaperone proteins within the organelle facilitate pulling the protein through and folding it once it s arrived EXAMPLE: Transport of unfolded protein in the mitochondria 3. Vesicular Transport Transport vesicles are small membrane enclosed compartments that move proteins throughout the cell Pinch off from one compartment and with another - EX: Leave the ER and fuse with the plasma membrane Internal environment can hold many different types of proteins or molecules - Soluble or membrane attached EXAMPLE: Vesicular transport from the ER to the Golgi and the plasma membrane Page 6
PRACTICE: 1. Which of the following is not a type of transport? a. Gated transport b. Organelle transport c. Vesicular transport d. Transmembrane transport 2. The nuclear pore is classified as which of the following types of transport? a. Gated transport b. Organelle transport c. Vesicular transport d. Transmembrane transport Page 7
CONCEPT: ER PROCESSING AND TRANSPORT Two types of ER import Co-translational import is the process of proteins into the ER as they re being translated ER signal sequence directs the mrna and ribosome to the ER during translation (located on N-terminus) - Signal recognition particle (SRP) recognizes the ER signal sequence - Signal recognition particle receptor (SRPR) is located on the ER and binds to the SRP Translocon is a pore in the ER membrane that binds SRP and SRPR to translocate the protein into ER - Uses GTP hydrolysis for energy - Signal peptidase cleaves the ER signal sequence off once it is inside the ER EXAMPLE: Co-translational import of a protein Post-translational import is the process of importing proteins into the ER they ve been translated The chaperone protein BiP helps pull the protein across the membrane and fold it once its inside ER-retention signals are located on the C-terminus and keeps proteins within the ER EXAMPLE: Posttranslational import Cytosol ER Lumen BiP Page 8
Inserting Proteins into the Membrane Single pass transmembrane proteins have one insertion in the Start transfer signal is a signal sequence which opens a translocon and the protein is translocated through it - Stop transfer signal signals to anchor the protein in the membrane - The ER signal sequence is then released from the translocon and cleaved off Start transfer signal can be on the N-terminus or located within the middle of the polypeptide chain EXAMPLE: Insertion of single pass transmembrane protein Single Pass Transmembrane Protein Stop Transfer Sequence Cytosol ER Lumen Start Transfer Sequence Signal Peptidase COOH NH 3 Multi pass transmembrane proteins have more than one insertion in the membrane Polypeptide chains contain multiple start and stop sequences - Order of sequence determines whether it is a start or stop sequence (the start sequence is first) - Multiple start transfer sequence initiate translocation at different sections along the polypeptide chain Page 9
EXAMPLE: Insertion of a multi pass transmembrane protein Multi Pass Transmembrane Protein Stop Transfer Sequence Start Transfer Sequence COOH NH 3 Cytosol ER Lumen ER Protein Modifications The ER is a major hub of protein Glycosylation of proteins occurs in the ER - Dolichol is a precursor oligosaccharide which is added to proteins for N-linked glycosylation - Oligosaccharides are important tags to mark the state of protein folding chaperons bind them Glycosylphosphatidyl-inositol (GPI) anchor is added to proteins destined for the plasma membrane - Can be easily cleaved off in order to release proteins into the extracellular environment EXAMPLE: Glycosylation in the ER Page 10
Protein disulfide isomerase assists with protein disulfide bond formation Unfolded protein response detects misfolded proteins - ER-associated degradation (ERAD) proteins recognize misfolded proteins and transports them to cytosol EXAMPLE: Unfolded Protein Response in the ER Page 11
PRACTICE: 1. Match the following term with its definition I. Co-translational import II. Post-translational import III. ER retention signal IV. Translocon A. Pore in the ER membrane that binds SRP and SRPR to translocate the protein into ER B. Signal sequence located on the C-terminus and keeps proteins within the ER C. Process of importing proteins into the ER as they re being translated D. importing proteins into the ER after they ve been translated 2. Which of the following is responsible for recognizing the ER signal sequence? a. Signal Recognition Particle b. Signal Recognition Particle Receptor c. Translocon d. Stop Transfer Sequence Page 12
3. A protein contains 5 start/stop transfer sequences. How many times will this protein cross the membrane? a. 2 b. 5 c. 10 d. 3 4. Glycosylation of proteins in the ER is associated with which of the following molecules or responses? a. Unfolded protein response b. Protein disulfide isomerase c. Glycosylphosphatidyl-inositol (GPI) anchor d. Dolichol Page 13
CONCEPT: GOLGI PROCESSING AND TRANSPORT Proteins Leaving the ER Proteins leaving the endoplasmic reticulum arrive at the Golgi complex (apparatus) Proteins are marked for exit by sorting signals, which sort them into special COPII vesicles (COPII protein coat) Only properly proteins can exit the ER for the Golgi - Chaperone proteins (BiP and calnexin) control folding - Improperly folded proteins are transported out of the ER into the cytosol for proteasome degradation Arrival at the Golgi depends on vesicle - Heterotypic fusion: membrane fusion from two different compartments (ER vesicle fusing with Golgi) - Vesicular tubular clusters: ER vesicles fuse together to create a big compartment that fuses with the Golgi ER resident proteins contain retrieval sequences that direct them back to the ER if they leave - KDEL sequence bind to KDEL receptors which direct them back to the ER via COPI vesicles (COPI coat) EXAMPLE: Types of arrival at the Golgi Golgi Vesicular Heterotypic Fusion Vesicular Tubular Clusters Endoplasmic Reticulum Nucleus Page 14
Golgi Structure The Golgi Complex (apparatus) has a structure made up of flattened membrane-enclosed sacs Cisternae are the flattened membrane enclosed stacks that make up the Golgi - The number of cisternae varies between 3-20 depending on the cell type The Golgi is ordered into three sections 1. The cis-golgi faces the endoplasmic reticulum and takes in proteins from the ER 2. The trans-golgi (TGN) faces the plasma membrane and is the location of protein exit 3. The medial-golgi sits between the cis and trans faces, and is the place where proteins are modified EXAMPLE: Structure of the Golgi complex Page 15
Glycosylation and Other Protein Modifications The Golgi complex is a major location where proteins are Glycosylation or the addition of carbohydrates occurs in two forms on proteins - N-linked glycosylation links an oligosaccharide to a nitrogen atom on an asparagine - O-linked glycosylation links an oligosaccharide to a hydroxyl group on a serine or threonine Protein glycosylation helps in protein folding and stability EXAMPLE: Two forms of glycosylation Terminal glycosylation is the final modification that removes carbohydrates. It occurs in the Golgi - N-terminal glycosylation occurs in the ER with removal of glucose and mannose sugars in the Golgi - Glycosyltransferases and glycosidases are the two main enzymes responsible for this modification Each modification occurs in a different cisternae within the Golgi - Functional differences between the three Golgi regions (cis, trans and medial) EXAMPLE: Protein modifications occurring in the Golgi apparatus Page 16
Golgi Maturation and Protein Transport Molecules move through the Golgi in two main ways Vesicular transport model: Golgi cisternae are stationary, and proteins move via vesicles between golgi stacks Cisternal maturation model: Golgi cisternae move upwards while maturing, disappearing due to vesicle budding - Replaced by the cisternae below it Evidence suggests proteins move using a combination of the two pathways EXAMPLE: Two methods of protein travel through the Golgi Protein transport occurs in two Anterograde transport: Moves from ER through Golgi and towards the plasma membrane Retrograde transport: Moves from the plasma membrane to the Golgi and then to the ER Golgi acts as a sorting hub: recognizes sorting sequences and sorts them into vesicles for transport - Protein receptors on the TGN bind to sorting sequences and trigger proper sorting and packaging Page 17
EXAMPLE: Anterograde vs. Retrograde Transport Plasma Membrane Retrograde Transport Golgi Anterograde Transport Nucleus Endoplasmic Reticulum PRACTICE: 1. Which of the following is not a method of Golgi transport? a. Retrograde transport b. Vesicular transport model c. Cisternal transport model d. Medial golgi transport Page 18
2. Which side of the Golgi faces the endoplasmic reticulum? a. Cis-Golgi b. Trans-Golgi c. Medial-Golgi 3. True or False: Each Golgi cisternae matures by moving upwards through the Golgi. a. True b. False Page 19
4. Which of the following transport moves molecules from the plasma membrane to the Golgi? a. Cisternal transport b. Vesicular transport c. Retrograde transport d. Anterograde transport Page 20
CONCEPT: VESICULAR BUDDING, TRANSPORT, AND COATS Overview Transport vesicles carry molecules between organelles and the plasma membrane Secretory pathway beings in the ER, moves to the Golgi, and ends at the cell surface Endocytic pathway beings at the plasma membrane, and brings molecules into different organelles EXAMPLE: Secretory pathway in the cell Vesicular Coats Many times, vesicles bud from organelles or other cellular structures surrounded by a coat There are three main types of vesicular coats - Clathrin coated vesicles travel between the Golgi and plasma membrane - COPI coated vesicles bud from the Golgi towards the ER - COPII coated vesicles bud from the ER EXAMPLE: Coated vesicle Page 21
Clathrin coats drive vesicular - Adaptor proteins bind the clathrin, and transmembrane proteins that are being transported - Cargo receptors are transmembrane proteins that capture soluble cargo - Dynamin is a cytoplasmic protein that assembles a ring around the neck and uses GTP to pinch it off EXAMPLE: Clathrin coated vesicle formation GTPases regulate recruitment of to the membrane - A coat protein binds to a cargo molecule or adaptor which triggers a GDP to GTP transition - GTP activated coat protein associates with the membrane and recruits more coat proteins - Rab proteins are GTPases that control specificity of coat proteins and specificity of transport - GTP form is tightly associated with membranes - Each vesicle contains unique combination of Rab proteins Page 22
EXAMPLE: Rab proteins in various organelles SNARE Proteins and Vesicular Fusion SNARE proteins are responsible for catalyzing membrane fusion and providing specificity to membrane fusion There are two types of SNARES which reside within the - T-snare has 2-3 target snares and resides on the target organelle. - V-snare is 1 protein that resides on the vesicle The two SNARES come together to form a four helix bundle (trans-snare complex) - Causes fusion of the vesicle to the target membrane To unravel the SNAREs energy from ATP is needed - NSF (N-ethylmaleimide sensitive factor) releases V and T SNARES using ATP hydrolysis Page 23
EXAMPLE: SNARE protein and vesicular fusion PRACTICE: 1. Which of the following is not a protein coat? a. COPI b. COPII c. COPIII d. Clathrin Page 24
2. Which of the following coats is used on vesicles that transport molecules from the Golgi to the ER? a. COPI b. COPII c. COPIII d. Clathrin 3. Vesicle fusion requires all but which of the following? a. T SNARES b. V SNARES c. Trans SNARE complex d. Rab GTP proteins Page 25
CONCEPT: TARGETING PROTEINS TO THE MITOCHONDRIA AND CHLOROPLASTS Proteins entering into the mitochondria and chloroplast have sequences that direct them to the organelle A chaperone protein (Hsc/Hsp70) carries the protein to the organelle - ATP hydrolysis removes the protein so it can pass through the TOM protein The TOM (TOC- for chloroplast) protein complex on the outer membrane recognizes a signal sequence - The protein is unfolded and led into the intermembrane space The TIM (TIC for chloroplast) protein complex on the inner membrane recognizes a second signal sequence - A H + gradient drives the passage of the protein through the TIM complex proteins within the organelle bind and help it refold EXAMPLE: Protein feeding through the TOM/TIM complexes to enter the mitochondria Hsc/Hsp70 1. ATP ADP + P i 2. TOM/TOC Cytosol H + H + H + H + H + H + H + H + H + 3. TIM/TIC Intermembrane space H + 4. H + Chaperone Protein Stroma Proteins are directed to many within the organelles There are many sub compartments within the mitochondria and chloroplasts - Signal sequences direct the protein to each compartments Some proteins need to insert into various organelle membranes - Inserts the same way as other transmembrane proteins EXAMPLE: Insertion into a thylakoid membrane Page 26
Stop Transfer Sequence Stroma Thylakoid Lumen Start Transfer Sequence Signal Peptidase COOH NH 3 PRACTICE: 1. True or False: Proteins must remain intact in order to cross the membrane and enter the mitochondria or chloroplast? a. True b. False Page 27
2. The energy to insert proteins through the TIM/TIC complex comes from what reaction? a. ATP hydrolysis b. H + gradient c. GTP hydrolysis d. Ca 2+ influx into the mitochondria or chloroplast Page 28
CONCEPT: LYSOSOMAL AND OTHER DEGRADATIVE PATHWAYS Lysosomal Characteristics and Sorting The lysosome contains many enzymes that intracellular and extracellular materials The lysosomal lumen is extremely acidic - ATP H + pump controls the acidity by pumping protons into the lysosome - Lysosomal membrane is protected from the acidity due to glycosylation on the lipids and proteins Lysosomal acid hydrolases are enzymes within the lysosomal lumen that breakdown materials - Around 50 types in each lysosome EXAMPLE: Lysosome with a variety of lysosomal acid hydrolases Proteins destined for the lysosome must contain the appropriate A mannose 6 phosphate tag is added onto lysosomal proteins in the Golgi - This tag is recognized by lysosomal receptors in the Golgi - Proteins with this tagged are sorted into transport vesicles destined for the lysosome EXAMPLE: Mannose 6-phosphate tag directing proteins to the lysosome Lysosome M6PR Page 29
Other Degradation Pathways Autophagy is the process of the cell eating itself by large molecules or organelles Autophagosomes form by enclosing old organelles with a double membrane - This large membrane fuses with the lysosome (autolysosome) for degradation - Also called macrophagy (microphagy is a smaller vessel with a single bilayer) EXAMPLE: Autophagosome and autolysosome formation Phagocytsosis is the process of the cell internalizing large particles from the extracellular environment for degradation Phagosomes form by enclosing the molecules with a membrane - This large membrane fuses with the lysosome (phagolysosome) for degradation EXAMPLE: Overview of Phagocytosis Page 30
PRACTICE: 1. Which of the following terms describes the cell eating itself? a. Lysosomal degradation b. Autophagy c. Phagocytosis d. Pinocytosis 2. The mannose 6 phosphate tag is added onto proteins for what region? a. Targets them to the lysosome for their function b. Targets them to the lysosome for degradation c. Targets them for autophagosomes d. Targets them to the cytoplasm Page 31
3. Macrophagy differs from microphagy in which of the following ways? a. Macrophagy degrades proteins and microphagy degrades small molecules b. Macrophagy is involved in cell death, microphagy is involved with taking things into the cell c. Macrophagy uses two membranes, microphagy uses one membrane d. Macrophagy uses autophagosomes, microphagy uses lysosomes Page 32
CONCEPT: ENDOCYTIC PATHWAYS Three types of endocytosis There are three methods of endocytosis which bring fluid or other molecules the cell Pinocytosis (cell drinking) brings fluid into cells Phagocytosis (cell eating) brings large molecules into cells - Phagosome fuse with lysosomes in order to degrade molecules Receptor-mediated endocytosis is a highly specific way to bring molecules into cells - Clathrin coated pits bind specific receptors which select cargo for entry (LDL and cholesterol) - Caveolae (small invaginations of the plasma membrane) can also specifically bring molecules into the cell EXAMPLE: Three types of endocytosis Page 33
Endosomal Sorting Endosomes are a large organelle sorting for a variety of materials entering and exiting the cell There are two types of endosomes: early and late - Late endosomes are a more mature form of early endosomes Multivesicular bodies form when invaginations from the endosomal membrane form internal vesicles - Carries these internalized vesicles to the lysosome for degradation EXAMPLE: Multivesicular body formation and transport to the lysosome Endosomes are slightly compartments which acts to release receptors from their cargo - Recycling endosomes carry receptors back to the plasma membrane - Cargo can travel to the lysosome or other organelles - Transcytosis occurs If cargo remains attached to the receptor and it travels to a different cellular spot - Cargo that remains bound to receptors ends up wherever the receptor ends up EXAMPLE: Recycling endosomes and transcytosis Recycling Endosome Endosome Transcytosis Page 34
Cholesterol (LDL) Uptake Receptor-mediated endocytosis is responsible for taking up from the blood stream Low-density lipoproteins (LDLs) are the main form of cholesterol found in the blood - LDL receptors are present on the plasma membrane When the cell needs cholesterol it makes LDL receptors and places them in the plasma membrane - These receptors then diffuse in the membrane until they come in contact with clathrin-coated pits - LDL receptors then bind LDL which allows for interaction with adaptor proteins - This triggers further formation of the clathrin coated pit and internalization of LDL cholesterol High blood cholesterol develops in individuals with mutations in the LDL receptor EXAMPLE: Diagram of LDL Uptake LDL LDL Receptor Clathrin Coated Pit Clathrin LDL Receptor Recylcing Separation of LDL from LDLR Page 35
PRACTICE: 1. Which of the following is not an endocytic pathway? a. Receptor-mediate endocytosis b. Pinocytosis c. Fluid uptake d. Phagocytosis 2. Which vesicle is responsible for carrying receptors back to the plasma membrane? a. Early endosomes b. Late endosomes c. Recycling endosomes d. Multivesicular bodies Page 36
3. High blood cholesterol develops when what happens to the LDL receptor? a. Too much LDL receptor is produced b. Too little LDL receptor is produced c. LDL binds the LDL receptor d. The cell takes in too much LDL in clathrin coated pits Page 37
CONCEPT: EXOCYTOSIS Exocytosis is the process of molecules outside of the cell Constitutive secretory pathway is the default pathway that operates continuously - Supplies the plasma membrane with newly synthesized lipids and proteins Regulated secretory pathway occurs in specialized secretory cells that produce a lot of a released molecule - Secretory vesicles store this molecule for later release - Bud from Golgi, but dock near the plasma membrane for release - Releases material in response to a chemical signal (calcium) - Common in nerve cells Microtubules vesicles to the cell surface EXAMPLE: Regulated secretory pathway in a neuron Page 38
Exocytosis occurs in four steps 1. Vesicle carrying cargo moves towards the plasma membrane - Polarized membranes have different domains - Vesicles are targeted to one domain via C-terminal sorting signals 2. Vesicle fuses with the plasma membrane 3. Vesicular contents are released into the extracellular environment 4. Membrane fusion causes the membrane to become larger - Endocytosis (molecules entering the cell) reduces membrane size EXAMPLE: Vesicular fusion steps 3. 2. 1. Page 39
PRACTICE: 1. Which of the following is not a pathway of exocytosis? a. Constitutive secretory pathway b. Regulated secretory pathway c. Continuing secretory pathway 2. When a vesicle fuses to the plasma membrane what happens to the lipids in the vesicles? a. They are released with the contents of the vesicle b. They are degraded c. They are incorporated into the plasma membrane Page 40
CONCEPT: PEROXISOMES Peroxisomes are organelles that contain chemical reactions Uses oxygen to produce hydrogen peroxide (H2O2) - Catalase is the enzyme that uses H2O2 is then used to oxidize other toxic molecules (ex: ethanol) Around 50 enzymes function in various biochemical pathways - Lipid synthesis including plasmogens (phospholipids with glycerol attached with a ether bond) - In plants it is the place of the glyoxylate cycle which converts fatty acids to carbohydrates - Beta oxidation (breakdown of fatty acids) occurs here EXAMPLE: Oxidizing toxic molecules using hydrogen peroxide Peroxisomes are in the ER - Bud off and fuse with other buds to form new peroxisomes SKL sorting signal exists on peroxin proteins that reside in the peroxisomes - Defects in import of perixosomal proteins can cause severe diseases (Zellweger syndrome) Page 41
PRACTICE: 1. What is the function of a peroxisome? a. Maintain acidity of the cell b. Contain toxic reactions c. Protein synthesis d. Protein glycosylation Page 42
CONCEPT: PLANT VACUOLE Plant vacuoles are large, enclosed vacuoles with many functions Functions for the plant vacuole include: - The membranes are acidified and can act similarly to lysosomes - Maintain turgor pressure to prevent cellular collapse - Regulate cytosolic ph through ATP dependent proton pumps - Stores nutrients and other solutes The plant vacuole is from a pro-vacuole (similar to an endosome) which matures to a vacuole - All vacuole components are produced in the ER, and transferred to the Golgi for processing - After processing, vesicles are released that eventually fuse to form the pro-vacuole - Tonoplast is the membrane that bounds the vacuole EXAMPLE: Overview of a plant cell Page 43
PRACTICE: 1. Which of the following is not a function of the plant vacuole? a. Maintain turgor pressure b. Regulate cytosolic ph c. Support protein transfer in the plant cell d. Store nutrients and solutes Page 44