Molecular Biology of the Cell

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Unformatted text preview: Lecture 1 Click to edit Master subtitle style Inner Life of the Cell • http://www.youtube.com/watch?v=BtZEqQ1cpmk Biology Department Goals Goal 1. Students will gain a knowledge of the key areas within the field of Biology. Goal 2. Students will develop skills in analysis and critical thinking through experimentation. Goal 3. Students will master basic laboratory skills. Goal 4. Students must gain an understanding of the relationship between science and society, primarily as this relationship pertains to ethical issues. Outline Monomer Polymer Protein Function • What are some examples of general biological functions carried out by proteins? Proteins • Functions – Support – Enzymes – Transport – Defense – Hormones – Motion Proteins • Amino acids are subunits of proteins. – Bond to a hydrogen atom, an amino group -NH2, an acidic group -COOH, and an R (remainder) group. v Peptide Bond - Covalent bond between two amino acids. Ø Peptide - Two or more amino acids bonded together. Ø Polypeptide - Chain of many amino acids joined by peptide bonds. H H2 N C CH2 CH2 CH2 CH2 NH3 + O H2 N O H C H O O Typical Amino Acids (See Fig. 2.23, Panel 3-1) Glycine Lysine H H2 N C CH2 O O Amino acids are subunits of proteins. Make up: 1. hydrogen atom, 2. an amino group -NH2, 3. an acidic group -COOH, 4. unique R-group /side chain. differs for each AA Phenylalanine 04_03_20 amino acids.jpg Structural Components of Protein Fig 3-2, Peptide Bond Formation • • • Carbon atom (●) from carboxyl group of one amino acid shares electrons with nitrogen (●) from amino group of second amino acid Condensation reaction Requires energy input Structural Components of Protein Fig 3-2, Structural Components of Protein • Folding of the Protein is important to the Function of the Protein AA side chains can be: – Negatively charged – Positively charged – Uncharged, polar – Nonpolar Fig 3-2, Noncovalent Bonds Help Proteins Fold Fig 3-5, Hydrogen Bonds Play Many Roles In Protein Folding Fig 3-7, Hydrophobic Residues Affect Folding in Aqueous Environments Fig 3-6, Disulfide Bonds Affect Protein Folding Fig 3-29, Secondary Structure: α Helix Fig 3-9, Secondary Structure: β Sheet Fig 3-9, Src is a Protein Containing Four Domains Fig 3-12, Variety of Shapes Seen in Protein Domains Fig 3-19, Comparison of DNA-Binding Domains From Yeast and Drosophila Proteins Bind to Other Molecules • Protein-protein binding – Multimeric proteins (tertiary structure) – Filamentous proteins – long fibers – Assembly of large structures v Ribosomes v Multienzyme complexes v Viruses Proteins Can Form By Association of Multiple Subunits Cro repressor protein: dimer of two identical subunits Hemoglobin: tetramer of two alpha globin and two beta globin chains Protein-Protein Binding Fig 3-26, MBOC4 Fig 3-22, MBOC4 Fig 3-31, MBOC4 Co-Factors / Co-Enzyme Vitamins/Minerals Co-Factor- Non-Organic Molecule Co-Enzyme- Organic , however not a protein Aides in enzyme binding activity/Protein folding Protein-Protein Binding • • • • Ligand: substance bound by a protein Enzymes: bind substrates Hormone receptors: bind hormones Antibodies: bind foreign molecules Protein Folding • 1. Primary structure: sequence of amino acids 2. Secondary structure: regular folding patterns – α helix – β strands, β sheets 3. Tertiary structure: overall 3-dimensional form of folded polymer 4. Quaternary structure: several polypeptides interact to form protein Protein domain: substructure of a polypeptide chain; compact, stable independently folded region • • • • Protein Folding Diseases • As proteins are being synthesized, chaperones bind and prevent incorrect interactions. – Mad cow disease is an example of a category of fatal brain diseases TSEs, that could be due to misfolded proteins. Thought question –Protein synthesis • Which do you think you will have a greater chance of surviving. – Prolong Hyperthermia – Prolong Fever Lecture 2 Click to edit Master subtitle style Outline Types of regulation (general) Feedback inhibition – Double-Reciprocal l Allosteric regulation l Covalent bonding of additional side chains Regulating Protein Activity 3 main categories of enzyme regulation 1. Feed back inhibition 2. Allosteric regulation 3. Covalent modification – Phosphorylation/dephosphorylation Irreversible Inhibitors Permanently bound to enzyme Nerve gas- affect nervous system Some heavy metals Not really an option for the cell!!! Enzyme Inhibition Reversible means of regulation is more common Two ways inhibitors work – competitive inhibition: binds with active site Competing with the substrate – non-competitive inhibition: binds with sites other than active site Competitive inhibitor Resemble the substrate and competes with the substrate for active site binding – (substrate analog) However it does not react or form the same bonds as the substrate. principle of many drugs Can better see and understand results if we use a different graph to show enzyme kinetics Double-Reciprocal Plot Hard to extrapolate data using MichaelisMenton plot- hard to find exact data points Lineweaver-Burk double reciprocal plot of enzyme kinetics allows us to extrapolate data due to its linear nature. Can find exact Vmax and Km Data is inverse form Double-Reciprocal Plot Double Reciprocal Plot Slope of line = Km / Vmax Km can be calculated from the X-intercept Vmax from the Y-intercept Inverse relationship velocity Km Vmax Lower [s] Higher A competitive inhibitor increase the Km but does not affect Vmax Decreases affinity for substrateraises Km inhibitor Y- intercept is unchanged X - intercept is changed Normal Lower Higher Non-Competitive Non-competitive inhibitor competes for a different site inhibitor (allosteric effector ) Decreases Vmax ( higher y-intercept); usually with no change in Km (x intercept) Normal Lower Higher Why does completive only effect only effect KM while non-completive only effect Vmax ? Lower V Non Competitive Competitive Normal Lower Km Higher Higher V Feedback Inhibition n where a product of the reaction inhibits an enzyme n Excess of the final product leads to inhibition. Feedback Inhibition n Why does is Z (last product in the series) inhibit the path way? Why not Y or X ? How do we reverse? n Negative feed back can be part of larger pathways and inhibit multiple pathways Allosteric regulation allo = different or other, steric = structure or state allosteric activator - where the a.a. binds to enzyme and changes active site to fit substrate allosteric inhibitor - where the a.i. binds to enzyme and changes active site to NOT fit substrate differs from non-competitive inhibition Allosteric enzymes may be either inhibited or activited by regulator substances Reaction rate is sigmoid curve verses curved Allosteric regulation Can be positive or negative Negative effector usually decreases Vmax Postive effector can affect either Vmax, Km or both Cells use Allosteric effectors to regulate reactions because that is the easiest way to control the enzyme Allosteric Regulation Fig. 3­12 Enzyme regulation covalent modification – calcium ions, phosphate, methyl, acetyl, groups or derivatives of nucleotides Act as additional side groups these all cause conformational changes – – Kinases add a Phosphate Review Types of regulation (general) Feedback inhibition – Double-Reciprocal l Allosteric regulation l Covalent bonding of additional side chains Lecture 3 Click to edit Master subtitle style Substrate concentration ? Test- 2 Two “short” tests • Membranes (2.5 lectures) - Ch11 v Structure Lipids Proteins • Transport (3.5 lectures) – Ch12 v v v Active Facilitative Co-transport STRUCTURE OF CELLULAR MEMBRANES Click to edit Master subtitle style • Why do we have membranes • What do they do Roles of membranes v Compartmentalization Site of important reactions Regulate communication and transport Cell-cell communication v v v Outline General Properties and Components • Membrane structure v Fluid mosaic model • Membrane components v v v Lipids Carbohydrates proteins • Membrane biogenesis Membrane Composition • Lipid ~ 50% • Protein ~ 50%; ranges from 25%-75% • Carbohydrate – minor component Glycoproteins Glycolipids • Values above for “average” membrane; composition of membranes varies v v What makes up a Membrane ? Lateral Diffusion of Proteins in Membranes Fig 10-39, Phospholipid Mobility • Lateral diffusion is easier than vertical- (flip-flop) • flip-flop requires an enzyme Asymmetric Structure of Membrane Cholesterol Phosphatidylethanolamine Phosphatidylcholine Glycolipid Phosphatidylserine Sphingomyelin Fig 10-14, Self Assembly of Amphipathic Lipids Cause of bilayer Hydrophilic polar heads face outside, and hydrophobic nonpolar tails face each other Fig 10-4, Membrane Lipids • Phospholipids • Cholesterol v v v Essential for animal cell membrane function Can be synthesized, not needed in diet Not found in bacterial membranes Membrane lipids • Fatty acids & lipids • fatty acids v v saturated unsaturated Phospholipid Structure Phospholipid= glycerol + 2 FA + Phosphate Fig 10-2, Sphingolipid • based on sphingosine (FA + aa serine) • mainly another FA + phosphate or some other polar group. chains form lipid rafts • Very long chain hydrocarbon Sterols • 4-multicarbon rings v v from Acetyl-CoA 15 or 30 C • Cholesterol v v v v The primary sterol in animal membranes also found in plants (much lower percentage), not in prokaryotes other organisms use other sterols for stabilization Cholesterol Function (4) v increase mechanical strength of membrane lowers membrane permeability reduces fluidity of membrane at HIGH temp (= more stable) increases fluidity at LOW temps v v v lowers transition temperature (the temperature at which the membrane “freezes” or “melts”) Membrane fluidity & permeability • Transition temperature is the Temp that the membrane crystalizes ("freezes") or undergoes PHASE TRANSITION v e.g. liquid butter to solid butter below transition temp lipids crystalize & REDUCES diffusion membranes NOT fully functional in this state v v Cells can change the transition temp lower trans. temp (adaption to cold) • animals lower trans. temp (adaption to cold) a) increase proportion of Unsaturation (increase the number of double bonds) b) increase cholesterol content • plants a) increase proportion of Unsaturation (increase the number of double bonds) • prokaryotes a) increase proportion of Unsaturation (increase the number of double bonds) b) decrease FA chain length v v v v v How would an organism adapt to warmer temperatures? Membrane biogenesis. • A. phospholipids originate in ER, v 1. plasma membrane a. transferred to Golgi via transition vesicles b. then exported to plasma membrane via secretory vesicles which fuse with the plasma membrane v v 2. nuclear membrane is connected to ER 3. mitochondria & chloroplasts much of the lipid is transferred to mitochondria & chloroplasts via soluble cytoplasmic proteins called lipid transfer proteins or exchange proteins. What makes up a Membrane ? Thought Question • Properties of a Membrane are determined by the structure of the lipid molecules. Predict what would happen to the lipid bilayer if the following were true (or happened) 1. Phospholipids (PL) have had only one hydrocarbon (HC) chain instead of two 2. HC chains were shorter (10 carbons) 3. All HC were saturated 4. All HC were unsaturated 5. Bilayer contained a mixture of two kinds of lipid molecule: One with two saturated HC tails and one with two unsaturated HC tails 6. Each lipid molecule is covalently linked v • • • • • • To molecule next to it Lecture 4 Click to edit Master subtitle style -- Monkeys & Peanuts Assumptions: – – – 1. monkeys are full and don’t eat peanuts 2. peanuts can readily be stuck back together 3. monkeys are just as likely to put the peanuts back in the shells as they are to take them out. Substrate concentration – Monkeys & Peanuts [S] = Concentration of peanuts(peanuts/m2) Time required per peanut: To find (sec/peanut) To shell (sec/peanut) Total (sec/peanut) Rate of shelling Per monkey (peanut/sec) Total (v) (peanut/sec) 9 1 10 0.10 1.0 1 Substrate concentration – Monkeys & Peanuts [S] = Concentration of peanuts(peanuts/m2) Time required per peanut: To find (sec/peanut) To shell (sec/peanut) Total (sec/peanut) Rate of shelling Per monkey (peanut/sec) Total (v) (peanut/sec) 9 1 10 3 1 4 1 3 0.10 0.25 1.0 2.5 Substrate concentration – Monkeys & Peanuts [S] = Concentration of peanuts(peanuts/m2) Time required per peanut: To find (sec/peanut) To shell (sec/peanut) Total (sec/peanut) Rate of shelling Per monkey (peanut/sec) Total (v) (peanut/sec) 9 1 10 3 1 4 1 1 2 1 3 9 0.10 0.25 0.5 1.0 2.5 5.0 Remember we started out with 10 monkeys total Substrate concentration – Monkeys & Peanuts [S] = Concentration of peanuts(peanuts/m2) Time required per peanut: To find (sec/peanut) To shell (sec/peanut) Total (sec/peanut) Rate of shelling Per monkey (peanut/sec) Total (v) (peanut/sec) 9 1 10 3 1 4 1 1 2 0.33 1 1.33 1 3 9 27 0.10 0.25 0.5 0.77 1.0 2.5 5.0 7.7 Thought Questions What is happening at low concentration in terms of enzyme binding and turnover, substrate concentration High concentration….. Need to Graph? [S] = X axis V (peanuts shelled/time) = Y Axis Substrate concentration Substrate concentration What does Vmax tell us about an enzyme? What does Km tell us about an enzyme? Why is this important ? – Why study Km and V-max both give information about efficiency of an enzyme Km tells about the effective working concentration – if the concentration is below the Km then the reaction is very inefficient – preferred substrates vs. alternate substrates V-max tells about "turnover" or how fast the enzyme can complete the reaction Can also tell a lot about inhibitors such as for control of the reaction or testing drugs Regulating Protein Activity 3 main categories of enzyme regulation 1. Feed back inhibition 2. Allosteric regulation 3. Covalent modification – Phosphorylation/dephosphorylation Irreversible Inhibitors Permanently bound to enzyme Nerve gas- affect nervous system Some heavy metals Not really an option for the cell!!! Enzyme Inhibition Reversible means of regulation is more common Two ways inhibitors work – competitive inhibition: binds with active site Competing with the substrate – non-competitive inhibition / Allosteric: binds with sites other than active site Competitive inhibitor Resemble the substrate and competes with the substrate for active site binding – (substrate analog) However it does not react or form the same bonds as the substrate. principle of many drugs Competitive Inhibitor 1940s – end of WWII Probenicid BenzylPenicillin / Penicillin G Do not get caught up in the chemical details Allosteric regulation allo = different or other, steric = structure or state Allosteric enzymes may be either inhibited or activated by regulator substances allosteric activator - where the a.a. binds to enzyme and changes active site to fit substrate – increases activity allosteric inhibitor - where the a.i. binds to enzyme and changes active site to NOT fit substrate - decreases differs from competitive inhibition Allosteric regulation Can be positive or negative Negative effector usually decreases Vmax Postive effector can affect either Vmax, Km or both Cells use Allosteric effectors to regulate reactions because that is the easiest way to control the enzyme Allosteric Regulation Fig. 3­12 Double-Reciprocal Plot Hard to extrapolate data using MichaelisMenton plot- hard to find exact data points Lineweaver-Burk double reciprocal plot of enzyme kinetics allows us to extrapolate data due to its linear nature. Can find exact Vmax and Km Data is inverse form Double-Reciprocal Plot Double Reciprocal Plot Slope of line = Km / Vmax Km can be calculated from the X-intercept Vmax from the Y-intercept Inverse relationship velocity Km Vmax Lower [s] Higher Allosteric Inhibitor – Non change in Km / Vmax is decreased Non Competitive Inhibitors – Km – increased/ no change to Vmax A competitive inhibitor increase the Km but does not affect Vmax inhibitor Y- intercept is unchanged X - intercept is changed Normal Lower Substrate Concentration Higher Non-Competitive Non-competitive inhibitor competes for a different site inhibitor (allosteric effector ) Decreases Vmax ( higher y-intercept); usually with no change in Km (x intercept) Normal Lower Higher Substrate Concentration Why does completive only effect only effect KM while non-completive only effect Vmax ? Lower V Non Competitive Competitive Normal Lower Km Higher Higher V Feedback Inhibition n where a product of the reaction inhibits an enzyme n Excess of the final product leads to inhibition. Feedback Inhibition n Why does is Z (last product in the series) inhibit the path way? Why not Y or X ? How do we reverse? n Negative feed back can be part of larger pathways and inhibit multiple pathways Enzyme regulation covalent modification – calcium ions, phosphate, methyl, acetyl, groups or derivatives of nucleotides Act as additional side groups these all cause conformational changes – – Kinases add a Phosphate Review Types of regulation (general) Feedback inhibition – Double-Reciprocal l Allosteric regulation l Covalent bonding of additional side chains Lecture 5 Click to edit Master subtitle style MEMBRANE TRANSPORT Click to edit Master subtitle style Confusing Thought Question of the week In one of my wife's nursing classes she learned that on treatment for Hyperkalemia (to high [K ] in blood/extracellular fluid) was and IV of glucose and insulin. The teacher said when this was given K just follows the glucose through the membrane. – – – A. what is wrong with this last statement. B why is hyperkalemia bad? C. Suggest a reason why this mechanism works. Transport needs to be unique for every cell and organelle within a 1. In simple diffusion and facilitated diffusion, molecules move from an area of high concentration (outside the cell) to an area of low concentration (inside the cell). It is implied there is greater energy outside of the cell. Why is there greater energy outside the cell? (Or: why is there greater energy when there is a greater concentration?) Concentration and Charge Differences Affect Movement of Molecules Fig 11-4, Rank the following substances from the most soluble (permeable) in a phospholipid bilayer to the least soluble (impermeable). O2, CO2, Na+, water, ethanol (CH3CH2OH), urea (H2NCONH2), glycerol (CH2OHCHOHCH2OH), 18-C fatty acid Relative Permeability of Synthetic Lipid Bilayer Fig 11-1, MBOC5 Passive Transport-General Passive transport --- includes simple diffusion (+ osmosis) and facilitated diffusion Characteristics in common energy source – concentration difference between the outside & inside of the cell l free energy released in diffusion. – – – direction of transport: with concentration gradient No energy expended by the cell (in form of ATP) Transport protein used in facilitated diffusion Carrier proteins Channel proteins Passive Transport Simple diffusion/Passive – no protein required Small, uncharged molecules Small, polar molecules (FACILITATED DIFFUSION) Fig 11-4, Rate of Transport by Carrier Protein What is going on ? Explain why the data for facilitated diffusion differ from that of simple diffusion Fig 11-7, Facilitated Diffusion – General transmembrane protein is a transporter but does not uses ATP Carriers or Channels - specific closely related molecule – – Ex: glucose transporter will also carry mannose, galactose BUT only D-isomers, not L-isomers bidirectional faster than simple diffusion Facilitated diffusion 1. Carrier Proteins binding of substance causes conformational change, shifting the "opening" to the other side. this shift also lessons affinity of transporter to substance and substance released. Interesting side note: Glucose transporter Glucose moves into the liver – Carrier proteins How does a liver cell keep from reaching the saturation point. What keeps glucose from moving back? Glucose transporter mechanism used to keep the concentration of glucose lower on the inside of the cell than on the outside: Cell converts glucose --> G-6-P (“glucose - 6 - phosphate”) G-6-P cannot pass through the transporter. Selectivity – Facilitated Transport 2. Ion Channel Proteins Plasma membranes of plant, animal cells Cell can control opening and closing of channels but movement is with concentration gradient – no direct contact with substrate Fig 11-20, (Ion) Channel Proteins Can be opened or closed to prevent transport Carrier Proteins vs. Channel Proteins What are the differences and similarities between the two Fig 11-3, Recently, researchers found that when E. coli cells are exposed to a hypertonic (high solute) solution, the bacteria produce a permease that can actively transport K+ into the cell. Of what value is the active transport of K+, which requires ATP? Active transport – General Characteristics direct or secondary direction of transport -- against concentration gradient or electrical potential gradient – unidirectional requires energy (= ATP or light) and is sensitive to metabolic poisons requires transmembrane protein can be saturated (i.e. there is a max rate) carriers are specific -- carry single or Ways Of Driving Active Transport Secondary Active transport Characteristics of both active and passive Fig 11-8, Direct Active Transport use pumps that are directly dependent on ATP (they hydrolyze ATP to ADP + Pi) Different types of “pumps”: Thought Questions The red blood cells (rbcs) in your body maintain internal concentrations of sodium & potassium ions that are significantly differently from those in blood plasma. However, these gradients of ion concentration can be abolished if isolated rbcs are treated in any of the following ways. Explain how the effectiveness of the treatment shows that the movement of Na and K are energy-requiring processes. (a) If isolated rbcs are held for an extended period of time at 37 α C in a medium without an energy source, they will eventually begin to leak K ions outward and Na ions inward. However, the outward movement of Na ions and inward movement of K ions can be restored by the addition of glucose to the medium. Na+ K+-ATPase pump – – 3 Na+ out, 2 K+ in result is NET charge across membrane (High conc. of Na+ outside) – – animals only (none in prokaryotes, fungi, plants) Na/K pump is a P-type (phosphate) Model For Na+/K+ Pump Action Fig 11-14, Reasons to Maintain Intracellular [Na+] Maintain membrane potential Regulate osmotic pressure by controlling intracellular [Na+] Na+ concentration gradient used for coupled active transport of other molecules - side note: three Na+ are pumped out while 2 K+ are pumped in - what does this do the net H+ pumps (V-type) ATPases- create ATP Use H+ movement to create ATP Sometimes called H+ pumps but that part of a larger class of pumps (V-type or vesicle type) Na/K pump is a P-type (phosphate) Can be reversed these pump H+ from cytoplasm into space enclosed by a vesicle membrane – – – lowers pH inside vesicle maintains neutral pH of cytoplasm examples of materials pumped in V-type pumps: sugars, amino acids, ions Thought Questions Ouabain is a cardiac glycoside that binds with the carrier proteins involved in the sodium-potassium pump and inhibits the protein's function (i.e. the pump stops). Describe what you expect to be the effects of ouabain on the transport of glucose into the cell How would you (a cell) move glucose from point A to point B Low [Glucose] High [Na] High [Glucose] Low [Na] Low [Glucose] High [Na] Example- Na+ Gradient Can Drive Glucose Transport Binding of glucose and Na+ is cooperative [Na+] higher outside of cell Glucose is more likely to bind carrier in A state Both glucose and Na+ enter cell more often than leave Secondary active transport – Energy for transport provided by Na+ gradient – Na+ gradient established by Fig 11-10, MBOC4 Secondary Active Transport Pumps Couples movement of one molecule with another- Coupled or Co - transport Secondary active transport? Indirectly uses concentration gradient created by active transport Still relies on simple diffusion. Directly does not require energy Types of Coupled Transport Remember two molecules are moving 1. One is moving with a concentration gradient created by active transport 2. The other is moving against the gradient (high conc.) (low conc.) Facilitated Transport Fig 11-9, Transport of Glucose Across Cells Fig 11-12, Passive vs. Active Transport (FACILITATED DIFFUSION) Fig 11-4, Confusing Thought Question of the week In one of my wife's nursing classes she learned that on treatment for Hyperkalemia (to high [K ] in blood/extracellular fluid) was and IV of glucose and insulin. The teacher said when this was given K just follows the glucose through the membrane. – – – A. what is wrong with this last statement. B why is hyperkalemia bad? C. Suggest a reason why this mechanism works. Lecture 6 Click to edit Master subtitle style PROTEIN SORTING / Trafficking Chapter 11 & 12 Click to edit Master subtitle style Test 2 - Outline Ø Chapter 10- Membrane Structure/Proteins Ø Chapter 11 – Transport (small molecules) Ø Chapter 12- Protein Sorting l l Protein Folding / Ubiquitin Pathway CFTR – Ubiquitin Paper l Test 2 October 14th. Relative Volumes of Intracellular Compartments A Cytosol B Endosomes C ER – rough cisternae D ER – smooth cisternae + Golgi apparatus E Lysosomes F Mitochondria G Nucleus H Peroxisomes 54 ? ? ? ? ? ? ? Ø Ø Cytosol occupies 54% of total volume in liver cell What compartment ranks next in proportion of cell volume occupied? Relative Volumes of Intracellular Compartments A Cytosol B Endosomes C ER – rough cisternae D ER – smooth cisternae + Golgi apparatus E Lysosomes F Mitochondria G Nucleus H Peroxisomes 54 1 9% 6% 1 22% ? 1 Ø Ø Cytosol occupies 54% of total volume in liver cell What compartment ranks next in proportion of cell volume occupied? Relative Amount of Intracellular Membranes A B C D E F G H Endosomes ER – rough & smooth Golgi apparatus Lysosomes Mitochondria Nucleus Plasma membrane Peroxisomes Secretory vesicles ? ? ? ? ? ? ? ? ? Ø What membrane listed has the greatest proportion of membrane in the cell? Relative Amount of Intracellular Membranes A B C D E F Endosomes ER – rough & smooth Golgi apparatus Lysosomes Mitochondria- outter Mitochondria- inner Nucleus Plasma membrane ? 35+16 7 ? 7 32 ? 2 0.4 ? Ø What membrane listed has the greatest proportion of membrane in the cell? G H Peroxisomes Secretory vesicles Advantages of Internal Membranes Segregation of cytoplasm for specialized functions l Some cellular reactions incompatible • Protein synthesis/degradation l synthesis in cytosol l degradation in lysosomes • Oxidative reactions in peroxisomes Protein Trafficking- Outline 1. Gated transport: nuclearpores act as special gates that actively transport specific macromolecules Chapter 12 2. Transmembrane transport: transport of specific protein across a membrane -A. mt and chloroplast -B. primarily into the ER Chapter 12 3. Vesicular Transport: a membrane enclose a protein and moves it to a particular location Takes care of the majority of protein trafficking issues Chapter 13 Nuclear Envelope and ER Ø 1. Gated transport: nuclearpores act as special gates that actively transport specific macromolecules Fig 12-9, 2. Transmembrane transport: transport of specific protein across a membrane - A. mt and chloroplast - B. primarily into the ER (more later) l usually as an unfolded protein- Eventually folded once enters organelle (Mt,Cp, ER). After enters ER protein are then transport by Vesicles to other organelles. l Not Encoded by Human Mitochondrial DNA: Ø RNA polymerase Ø RNA processing and modifying enzymes Ø DNA replication enzymes Ø Ribosomal proteins Ø Some enzymes associated with CAC cycle Ø Main Point Not all proteins needed by the Mt are encoded on the MT DNA Protein Transport to: Ø Mitochondria l l Ø Chloroplasts l l l l Post-translational terminal signal peptide Signal peptide cleaved Energy required l l Post-translational terminal signal peptide Signal peptide cleaved Energy required Both Requires Translocator Protein Import By Mitochondria Protein Made in cytosol and then transport – (Post Translational) ØFig 12-26, The Postage Stamp Signal Peptide (Sequence) or Signal Patch Fig 12-8, Genetic Engineering to Study Signal Peptides Panel 12-1, MBOC Typical Signal Sequences Function of signal peptide Import into ER Return to ER Import into mitochondrion Import into plastid Example of Signal Sequence +H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-LeuVal-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-GluGln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln----Lys-Asp-Glu-Leu-COO+H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-ArgPhe-Phe-LysPro-Ala-Thr-Arg-Thr-Leu-Cys-SerSer-Arg-Tyr-Leu-Leu+H3N-Met-Val-Ala-Met-Ala-Met-Ala-Ser-LeuGln-Ser-Ser-Met-Ser-Ser-Leu-Ser-Leu-Ser-SerAsn-Ser-Phe-Leu-Gly-Gln-Pro-Leu-Ser-Pro-IleThr-Leu-Ser-Pro-Phe-Leu-Gln-Gly- Table 12-3, MBOC Ø. Endoplasmic Reticulum (ER) Ø (endo= within; -reticulum,= a network) Ø A. Structure l General: nuclear membrane, ER, Golgi all same (interconnected) system l 1. single membrane continuous w/ nuclear membrane 2. shape: network of mostly flattened sacs • • • a. sacs = cisternae (-na) 1) inside = cisternal space or ER lumen 2) divides cytoplasm into 2 parts Protein Trafficking 1. Gated transport: nuclearpores act as special gates that actively transport specific macromolecules 2. Transmembrane transport: transport of specific protein across a membrane -A. mt and chloroplast -B. primarily into the ER 3. Vesicular Transport: a membrane enclose a protein and moves it to a particular location Takes care of the majority of protein trafficking issues - General: nuclear outer membrane, ER, Golgi all same membrane system Endomembrane System Ø What are the structural and functional differences between rough and smooth endoplasmic reticulum? (RER/SER) l List the functions of each Ø What are the most important similarities between these compartments? Smoother ER Functions? 1. membrane biogenesis 2. Steroid Hormone synthesis 3. Drug Detoxification 4. Glycogen catabolism 5. Ca storage (muscle) A. Membrane Biogenesis Ø Ø Usually minor component 1. phospholipids originate in ER, l A. plasma membrane • • a. transferred to Golgi via transition vesicles b. then exported to plasma membrane via secretory vesicles which fuse with the plasma membrane l l B. nuclear membrane is connected to ER C. mitochondria & chloroplasts • much of the lipid is transferred to mitochondria & chloroplasts via soluble cytoplasmic proteins called lipid transfer proteins or exchange proteins. 2. Synthesize lipid-based compounds, e.g. steroid hormone biosynthesis • ex: male and female sex hormones 3. Detoxify lipid-soluble compounds • • • hydroxylation reactions (= addition of -OH) liver common tissue for detoxification modifies lipid soluble compounds to make water soluble; then excreted in urine Smooth ER/ Example 3. Drug Detoxification l drug detoxification example: Phenobarbital • • a) injection of phenobarbital results in rapid increase in a mono-oxygenase (a barbituratedetoxifying enzyme) in liver. -hydroxylation of drug makes it more soluble in water. Water soluble compounds are more easily “flushed” by the body. b) followed by a dramatic increase in smooth ER • drug detoxification (cont.)/ Smooth ER c) effect is that a greater amount of drug needed for same effect d) the monooxygenase that is induced by phenobarbital ALSO hydroxylates other drugs (some useful) (1) e.g. antibiotics (narcotics, steroids, anticoagulants) Thought Question How could widespread phenobarbital use in a population affect our ability to control disease? 4. Glycogen catabolism one function of liver is to convert glycogen glucose l 1) in liver, glucose is stored as glycogen: 2) converted to glucose to keep blood sugar concentrations level lycogen b glucose-1-P Glucose-6-P l 1 2 • but, membranes generally impermeable to phosphorylated sugars 3 l 3) in ER: G-6-P (glucose-6phosphatase) Glucose + Pi • then glucose can be transported into blood 5. Calcium Storage l Requires an ATP- dependent Ca pump (active process) Sarcoplasmic reticulum found in muscle cells • • l Type of ER that specifically stores CA Only in certain types of cells Protein Trafficking 1. Gated transport: nuclearpores act as special gates that actively transport specific macromolecules 2. Transmembrane transport: transport of specific protein across a membrane -A. mt and chloroplast -B. primarily into the ER 3. Vesicular Transport: a membrane enclose a protein and moves it to a particular location Takes care of the majority of protein trafficking issues - Lecture 7 Click to edit Master subtitle style STRUCTURE OF CELLULAR MEMBRANES Click to edit Master subtitle style Outline • Membrane Biogenesis • Membrane Carbohydrates • Membrane Proteins v v Integral Peripheral • Thought Questions Membrane biogenesis. • A. phospholipids originate in ER, v 1. plasma membrane a. transferred to Golgi via transition vesicles b. then exported to plasma membrane via secretory vesicles which fuse with the plasma membrane v v 2. nuclear membrane is connected to ER 3. mitochondria & chloroplasts much of the lipid is transferred to mitochondria & chloroplasts via soluble cytoplasmic proteins called lipid transfer proteins or exchange proteins. Membrane biogenesis • B. Proteins v a. proteins bound for plasma membrane, mitochondria & chloroplasts are made in the rough ER. b. The terminal end of the mRNA codes for a "signal mechanism" that is the "address" for the proteins be they bound for the p. membrane or organelle. v Membrane Carbohydrates Glycocalyx: carbohydrate-rich peripheral zone at cell surface Glycolipids: carbohydrate covalently joined to lipid in extracellular face Glycoprotein: carbohydrate covalently joined to side chains of protein; exposed on extracellular face Membrane Proteins Functions of Membrane Protein Transport needs to be unique for every cell and organelle within a cell Proteins • Two main classes of membrane proteins v peripheral & integral Integral- imbedded in hydrophobic interior - mono layer or bilayer (trans-membrane) v v peripheral held in place by hydrogen bonds On outside surface of membrane Does span into the hydrophobic interior Freeze-Fracture Splits Membrane, Proteins Associate With E or P Face Protein Arrangements in Membranes Sequence of peptide determines where in the membrane it will be found Determines shape Membrane spanning region Fig 10-17, Structure of a typical peripheral Amino acid sequence acts as and anchor Integral Membrane Proteins • Peptide bonds are polar • Interior of lipid bilayer is • How can proteins cross non-polar (hydrophobic) the hydrophobic environment within the lipid bilayer? Fig 10-19, Structure of a typical integral protein primary structure: hydrophobic aa are clustered in segments containing 20-25 residues Can form alpha-helices or beta sheets (barrels) v v v Can form pore protein Proteins Often Form Alpha-Helix to Cross Lipid Bilayer v the alpha-helices have nonpolar middles and polar ends Fig 10-19, β Barrels Cross Lipid Bilayers Integral proteins can form pores that allow molecules to move in and out of the cell Fig 10-21, Groups of Alpha-Helixes Cross Lipid Bilayer Integral proteins can form pores that allow molecules to move in and out of the cell Lateral Mobility of Proteins Can Be Restricted Fig 10-43, Some Proteins Restricted to Apical or Basal Domain of Cells Fig 10-41, Thought Question • Properties of a Membrane are determined by the structure of the lipid molecules. Predict what would happen to the lipid bilayer if the following were true (or happened) 1. Phospholipids (PL) have had only one hydrocarbon (HC) chain instead of two 2. HC chains were shorter (10 carbons) 3. All HC were saturated 4. All HC were unsaturated 5. Bilayer contained a mixture of two kinds of lipid molecule: One with two saturated HC tails and one with two unsaturated HC tails • • • • • Acholeplasma laidlawii is a small bacterium-like organism that is unusual because it uses whatever fatty acids are available in the environment for constructing its plasma membrane. When grown in a medium enriched with saturated fatty acids, A. laidlawii is forced to construct membranes with an abnormally high content of saturated fatty acids. When grown in a medium enriched with unsaturated fatty acids, A. laidlawii constructs membranes predominately of unsaturated fatty acids. PROBLEM. An experiment was conducted by growing A. laidlawii on two different media. Group S was grown on a medium rich in stearate, a saturated fatty acid; Group O was grown on a medium rich in oleate, an unsaturated fatty acid. Figure 2 is a graph of data collected from differential scanning calorimetry of the plasma membranes from the two colonies. Which of the peaks represents Group S and which peak represents Group O. Explain your answer. unsaturated fatty acid saturated fatty acid Relative Permeability of Synthetic Lipid Bilayer Fig 11-1, Concentration and Charge Differences Affect Movement of Molecules Fig 11-4, 1. In simple diffusion and facilitated diffusion, molecules move from an area of high concentration (outside the cell) to an area of low concentration (inside the cell). It is implied there is greater energy outside of the cell. Why is there greater energy outside the cell? (Or: why is there greater energy when there is a greater concentration?) Test 1 B lick to edit Master Cefore Corrections subtitle style ...
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This note was uploaded on 04/20/2010 for the course BIOL 4064 taught by Professor Dr.reyna during the Fall '09 term at Ouachita Baptist.

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