Exam 3 Lecture Notes

Chapter 11: Cell Membranes

Membrane Structure

All cells have them.

• Prokaryotes: 1 membrane → the cytoplasmic membrane (plasma membrane)

• Eukaryotes: Multiple membranes → cytoplasmic membrane + all the various internal membranes (membrane-bound organelles)

General Properties

1. Fluid

   • Able to change shape very easily; flexible. Individual components that make up the membrane are usually free to move around in various ways.

   • Ex: Fibroblasts - Assume these really extreme angles.

   • Self-annealing.

2. Selectively Permeable

   • Selective about which molecules they allow to permeate the membrane.

   • How they maintain certain chemical environments.

3. Mosaics

   • Mixtures of different kinds of molecules (lipids, proteins, carbohydrates, etc.)

   • Ex: Human RBC, plasma membrane

     • ~43% lipids by weight

     • ~49% proteins by weight

     • ~8% carbohydrates by weight

   • Ex: Human neuron, plasma membrane

     • ~79% lipids

     • ~18% proteins

     • ~3% carbohydrates

4. Asymmetric

   • The two halves of the bilayer are chemically distinct; two layers with different compositions.

     • Cytoplasmic side (inner side)

     • Non-cytoplasmic side (outer layer)

       • AKA the luminal side

       • AKA the extracellular side

Components of Cell Membranes

1. Phospholipids

   • Primary structural components

   • Amphipathic (partially hydrophilic and partially hydrophobic)

     • Head: choline, phosphate, glycerol, polar, charged; Hydrophobic: Fatty acid chains, nonpolar, uncharged.

   • Spontaneously form a bilayer in water

     • If enough of them, they will seal off and form a ring (artificial cell membranes/liposomes).

   • Usually free to move around in various ways within one half of the bilayer

     • If they have any thermal energy: Flexion, which is the movement of the tails

     • Rotation is rotating on their axis/tails spin on head

     • Lateral Diffusion = drifting

     • Flipping = Rarely occurs, but means a phospholipid could jump from one half to the other half of a bilayer

   • New phospholipids are made in the luminal space of the endoplasmic reticulum

     • “Scramblases” - Scramble, or randomly distribute, the phospholipids from one monolayer to another

   • Golgi apparatus is where phospholipid asymmetry is generated

     • “Flippases” - Catalyzes the transfer of specific (scrambled) phospholipids to the cytosolic monolayer

   • One fatty acid hydrocarbon tail is straight (saturated) and one is kinked or bent (unsaturated)

     • Saturated: No double bonds (flexible)

     • Unsaturated: Have one or more double bonds (rigid, more inflexible) which causes kinks

       • This reduces/eliminates the possibility of “phase separation”

         • If both tails were saturated (straight), there would be less fluidity but the membrane would be easier to freeze (lose transport capacity and ability to transport vesicles).

         • If both tails were unsaturated (kinked), the membrane would be more fluid or even too flexible. It would also be harder to freeze.

         • Overall, this causes congregations/patches of phospholipids that freeze unevenly.

   • Ex: phosphatidylcholine

     

2. Glycolipids

   • Secondary structural & functional components

   • Ex: Galactocerebroside - In neuron plasma membranes

     

3. Cholesterol

   • Secondary structural component in animal cell membranes mainly

     • Affects membrane fluidity (more cholesterol = less flexible; less cholesterol = more flexible)

     • Able to relieve tension in the membrane by flipping

       • This is why cholesterol is so abundant in animal cells and not so much in plant cells—animal cells are exposed to much more extreme bending and movement

     

4. Proteins & Glycoproteins

   • Primary functional components (not so much structural)

   • Integral Membrane Proteins

     • Proteins that are integrated directly into the phospholipid bilayer

     • Transmembrane proteins

       • Span across/embedded in both halves of the bilayer

     • Membrane-associated proteins

       • Embedded in only one half of the bilayer

     • Lipid-linked proteins

       • Covalently attached to lipids that are part of one half of the bilayer

   • Peripheral Membrane Proteins

     • Noncovalently associated with integral membrane proteins

     • Signal transduction proteins and kinases

   • Functionally:

     • Transport

     • Receptors (hormone, cytokine, growth factor)

     • Enzymes (in mitochondria involved in cellular respiration)

     • Recognition

   • Up to 50% of membrane by weight

     

5. Carbohydrates

   • Attached to membrane lipids (glycolipids) + attached to membrane proteins (glycoproteins) → form the glycocalyx (outer layer of carbohydrates)

   • Noncytoplasmic side only (= extracellular side = lumenal side)

   • Attached to membrane lipids and proteins in the ER and the Golgi (done by enzymes)

   • Functions: Protection, lubrication, adhesion (helping cells stick to things or other cells), communication, cellular migration, etc.

   • Ex: Lymphocyte

     • Has extremely large nuclei

Membrane Proteins

Membrane Domains

• Portion of an integral membrane protein that is actually embedded in the bilayer; consists of all or mostly hydrophobic amino acids

  • Ex: Single alpha helix

    • Monolayer - one half of bilayer

    • Transmembrane domain - span both halves of bilayer (~19 amino acids)

      • Hydrophobic side chain amino acids have to face outward

      • Includes many receptors, enzymes, and recognition proteins

        • Ex: mHCI - Major histocompatibility complex; almost no chance that 2 people have the same one

        • Ex: mHCII - Heterodimer (two different subunits/alpha and beta); disulfide bonds facing outward to help hold shape of molecules together

        • Ex: CD4 and CD8 - Co-receptor on T cells (CD4) and activated t-lymphocytes when you get an infection. CD8 is a heterodimer with disulfide bonds anchoring to the plasma membrane

        • Ex: Golgi sialyltransferase - Enzyme embedded in the Golgi membrane by way of an alpha helix that adds sugars to proteins in the Golgi

  • Multiple alpha helices

    • Includes many in ion channels, receptors, and membrane-embedded enzymes

    • Often ½ of each alpha helix is hydrophobic and ½ is hydrophilic

      • Ex: bacteriorhodopsin-7 transmembrane alpha helices

        • Light absorption + proton pumping (photosynthesis in Halobacteria, more specifically Halobacterium salinarum).

      • Ex: G-Protein-coupled Receptor

        • Large family of receptors for cytokines, hormones, neurotransmitters, pheromones, etc… all have 7 transmembrane alpha-helical domains

          • β-adrenergic receptor = epinephrine receptor = adrenaline receptors

        • Smell and taste receptors

        • Opsins + rhodopsins (vision)

          • Ex: Voltage-gated Na⁺ ion channel involved in action potential in neurons (24 transmembrane alpha helices)

• One or more “rolled sheets” (β-barrels)

  • Ex: Porin in outer membranes of gram-negative bacteria + outer membrane of mitochondria

Kyte-Doolittle Hydropathy Plot

• Method to identify membrane domains in membrane proteins; graph of amino acids at each position vs. hydropathy score

• Hydropathy Score:

  • More positive = more hydrophobic

  • More negative = more hydrophilic

• Each position represents an amino acid

• Each score above 0 is hydrophobic; each score less than 0 is hydrophilic

• LSS = Leader Sequence

Acetylcholine

• Four transmembrane alpha helices (M1, M2, M3, and M4)

• LSS is hydrophobic

Chapter 12: Membrane Transport

Intracellular vs. Extracellular Environment

Phospholipid bilayers are selectively permeable.

• Small, hydrophobic molecules are able to cross the membrane very quickly and easily

  • O₂, CO₂, N₂, benzene

  • Makes cell respiration easy as it does not have to have any specific dedicated carrier or transporter due to the concentration gradient created

• Small polar (partial charge) molecules are able to cross the membrane easily on their own, just takes a little more time to wedge through the phospholipids

  • H₂O, glycerol, ethanol

• Larger (3 carbons or more) polar molecules are not able to cross on their own because they are bulky and polar (hydrophilic)

  • Amino acids, glucose

• Ions + Charged molecules do not cross on their own

  • H⁺, Na⁺, HCO₃^-, K⁺, Ca²⁺, Cl^-, Mg²⁺, amino acids, nucleotides

Types of Membrane Transporters

1. Channel Protein - Creates a hydrophilic pore to allow ions to move in or out of the cell

   • Passive only → Facilitated diffusion (high to low concentration through the hole)

   • Selective based on size and charge

   • Constitutive (always open) and Gated (open or closed) channels

     • Voltage-gated ion channels (open/close based on charge/electrolyte balance or imbalance)

     • Ligand-gated

     • Mechanically-gated

   • Ex: K⁺ ion channels

     • Most common type

     • Present in most cell types

     • Many subtypes

       • K⁺ leak channels - Constitutive; help maintain resting membrane potential

       • Ca²⁺ Gated - Open only in response to high levels of [Ca²⁺]

       • Voltage-Gated K⁺ Channel - Open in response to changes in resting membrane potential

   • Ex: Voltage-Gated Na⁺ Channels

     • Help propagate action potential in neurons

2. Carrier Proteins

   • Behave like enzymes

     • Michaelis-Menten Graph: the ½ V-max on the y-axis (rate of transport) corresponds with the K_m on the x-axis (concentration of transported molecule).

     • Direct diffusion across the membrane: Straight line (/)

   • Passive (work down a concentration gradient) or Active (needs energy, forcing an ion or molecule against a concentration gradient)

     • 3 Types:

       • Uniporter - Transport 1 type of ion or molecule in one direction either into or out of the cell

       • Symporter - Transporting two types of ions or molecules in the same direction simultaneously

       • Antiporter - Transporting 2 types of ions or molecules in opposite directions

     • Sources of energy for active transport:

       • ATP hydrolysis, concentration gradient (couple the downhill transfer of a molecule or ion to the uphill transfer of another molecule or ion; downhill process endergonic and uphill process exergonic; “coupled transport”), sunlight (bacteriorhodopsin)

   • Ex: Na⁺/K⁺ Pump

     • ATP-driven active antiporter

     • Ouabain binds to this pump to prevent K⁺ binding

     • In cytoplasm membranes of all animal cells (including humans)

     • Account for ~30% of all ATP use in animal cells

     • For every ATP molecule they hydrolyze, they pump 3 Na⁺ ions out of the cell and 2 K⁺ in

       • Generate Na⁺ gradient (High [Na⁺] outside the cell), K⁺ gradient (High [K⁺] inside the cell), and electrochemical gradient (“resting membrane potential”; positive on outside, negative on inside; ~70 mV difference)

       • Other carriers can tap into these gradients, which is how we get “action potential”

         • Temporary rapid depolarization, followed by a repolarization

     • Help animal cells regulate osmotic pressure, since they don’t have cell walls

   • Ex: Glucose Transporters (GLUTs); ~15 types

     • Passive glucose uniporter

       • Takes advantage of a preexisting glucose concentration to passively bind to those glucose molecules and transport glucose down a concentration gradient

     • Gradient-Driven Active Glucose Symporters

       • Transports glucose against the concentration gradient

       • Transports Na⁺ down its concentration gradient at the same time

   • Ex: Ca²⁺ Pump

     • ATP-driven active uniporter

     • Plasma membranes of eukaryotic cells; pump Ca²⁺ out of the cell

     • Animals muscle cells = in sarcoplasmic reticulum pump Ca²⁺ into lumen of S.R.

       • Keeps Ca²⁺ in cytoplasm low

   • Ex: Bacteriorhodopsin

     • Light driven active transporter

     • 7 transmembrane alpha helices

     • Plasma membrane of Halobacteria (archaea)

     • Performs light reactions of photosynthesis

     • Absorbs green light; transmits purple light

     • Pumps H⁺ out of the cell

       • Creates H⁺ gradient used by ATP synthases to make ATP

Summary of Action Potential

Action Potential - Electrical impulse that neurons use to rapidly deliver messages to the body; change in the neuron’s “resting membrane potential”.

1. Na⁺/K⁺ pumps in all animal cells generate Na⁺ gradient (high on outside, low on inside), a K⁺ gradient (high on inside, low on outside), and electrical gradient.

2. Neurons tap into the energy of these gradients to generate action potential.

3. Neuron stimulated by pressure, sound waves, light waves, chemicals, and a variety of initial stimuli → Gated passive Na⁺ channels in plasma membrane of the neuron open up.

4. Na⁺ diffuses into the cell → Depolarization begins (#1 on graph)

5. Threshold: ~ -50 mV. If threshold is not reached, depolarization will subside (failed initiations spot on graph)

6. If threshold is achieved, voltage-gated Na⁺ channels open → more Na⁺ rushes into the cell; membrane completely depolarizes (up to +35-40 mV) (#2 on graph)

7. Voltage-gated Na⁺ channels close after ~0.5 ms

8. Gated Passive K⁺ channels open → K⁺ diffuses out of the cell and repolarization occurs (#3 on graph)

9. ~5 ms must pass before gated passive K⁺ channels can be reactivated → “refractory period”

   • Gated K⁺ cannot be activated during this period. The purpose of this 5 ms delay is to trigger the propagation of action potential (self-propagating wave of depolarization) by triggering the opening of Na⁺ voltage-gated channels. By the time the refractory period has elapsed, the wave is further down and the Na⁺ has already been pumped out. This makes sure that the action potential goes in one direction.

10. Na⁺/K⁺ pumps restore resting membrane potential (#5 on graph)

11. Na⁺ which diffuses into cell at step 6 (#2 in graph) diffuses laterally and triggers neighboring voltage-gated Na⁺ channels to open → Propagates action potential along the length of the axon or dendrite.

12. Self-propagating wave of depolarization

• The dashed line on the -70 is the resting membrane potential

• The line on ~ -50 is the threshold

• Dashed line at top of graph

• Y-axis says ‘membrane potential (mV)’ instead of voltage (mV)

• Depolarization begins at #1 in the graph (step 4)

• Add #5 (for step 5) to failed initiations spot

Chapter 14: Energy Generation in Mitochondria + Chloroplasts

Mitochondria

• A typical eukaryotic cell contains ~1000 mitochondria

• May change shape + change location in a cell

• Do not have the ability to move on their own, so the cell itself moves it around (microtubules, motor vesicles)

• Reproduce inside cells by binary fission

• Contains two membranes

  • Acquired the second when they were originally engulfed by endosymbiosis

  • Outer membrane - smooth

  • Inner membrane - highly folded into structures called “cristae”

• Contain 4 “compartments”

  1. Mitochondrial Matrix (Inner Compartment)

     • Contains 67% of total mitochondrial protein

       • Enzymes for citric acid cycle (where it takes place; includes pyruvate dehydrogenase complex

       • Enzymes for fatty acid oxidation (digestion of fats)

       • Enzymes for amino acid oxidation (converting them to energy)

       • Where mitochondrial DNA, mitochondrial ribosomes, and mitochondrial tRNAs are located

  2. Inner Mitochondrial Membrane

     • Folded into “cristae” to increase surface area for packing in more proteins and enzymes

     • Impermeable to most molecules (if a molecule is not small and hydrophobic, it is not going to get across the membrane on its own)

     • Contains ~21% of total protein in mitochondria

       • Enzymes for oxidative phosphorylation (where chemiosmosis takes place) — includes proteins making up the ETC, the proton pumps, and ATP synthases.

     • Carrier proteins (for pyruvate, ADP, inorganic phosphate; ATP out)

  3. Outer Membrane

     • Contains ~6% of total mitochondria protein

     • Contains porin (channel formed by a beta sheet that rolled over on its side for a wide diameter hole. The holes are embedded in the outer membrane to allows things like pyruvate, ADP, and ATP out/in of the mitochondria). It is permeable to most small/medium size molecules.

     • Contains Bax and Bcl. 2 proteins → Involved in triggering apoptosis (cell suicide)

  4. Outer Compartment (Inter-membrane Space)

     • Contains ~6% of total mitochondria protein

     • Similar composition to cytoplasm

Oxidative Phosphorylation (= chemiosmosis)

• Indirect method of making ATP

• Takes place in inner mitochondrial membrane

1. NADH + FADH₂ deliver high energy electrons to the ETC in inner mitochondrial membrane

   • Modified nucleotide

   • High-energy electron carriers

     • Come from glycolysis → 2 NADH per glucose

     • Citric acid cycle → 8 more NADH + 2 FADH₂ per glucose

     • Fatty acid oxidation (in mito. matrix) → 1 NADH + 1 FADH₂ + 1 acetyl-CoA per each 2C unit on a fatty acid chain

     a.) NAD⁺ = nicotinamide adenine dinucleotide

     • Coenzyme in all cells

     • Carries 2 e^- + 1H⁺

       • NAD⁺ (oxidized) → NADH (reduced)

     • Delivers 2 e^- to NADH dehydrogenase (1^st component of ETC)

     b.) FAD = Flavin adenine dinucleotide

     • Coenzyme in all cells

     • Carries 2 e^- + 2 H⁺

       • FAD (oxidized) → FADH₂ (reduced)

     • Delivers 2 e^- to ubiquinone (2^nd component of ETC)

2. ETC consists of 3 large complexes + 2 mobile e^- carrier (~40 protein subunits)

   • Complex I = NADH Dehydrogenase Complex

     • e^- bind to Fe at “Fe-S” complex (“electron cage”) attached to 4 cysteines (Fe³⁺ → Fe²⁺)

     • Pumps H⁺ across the inner mitochondrial membrane to build up proton concentration gradient

   • Mobile carrier I = ubiquinone = Q = “coenzyme Q” = “CoQ10”

     • Not a protein

     • Carries 2e^-

   • Complex II = Cytochrome C Reductase Complex (= cytochrome b-c complex)

     • e^- binds to Fe in heme group (nonprotein Fe + porphyrin ring) attached to methionines (Fe³⁺ → Fe²⁺)

     • Pumps H⁺ across the membrane

   • Mobile Carrier II = cytochrome C

     • e^- binds to Fe in heme group (Fe³⁺ → Fe²⁺)

     • Highly conserved in evolution

   • Complex III = Cytochrome C Oxidase Complex

     • Binds 4 e^- at a time (2 Fe in heme group + 2 Cu) (Fe³⁺ → Fe²⁺) (Cu²⁺ → Cu⁺)

     • Transfers e^-’s to final electron acceptor (aerobic respiration) = O₂ → H₂O

     • Pump H⁺ across the membrane

3. e^-’s move automatically through the ETC

   • High energy (low stability) → Low energy (high stability)

4. ATP Synthases tap into H⁺ gradient across membrane to indirectly make ATP (chemiosmosis)

   • Efficient because each time the electrons are transferred, they are going into a more stable state

5. Symporters in the inner mitochondrial membrane tap into H⁺ gradient to move pyruvate + inorganic phosphate into the mitochondrial matrix

Drugs that Block ETC

1. Rotenone (pesticide/piscicide) + amytal (amobarbital = a barbituate = tranquilizer “truth serum”)

   • Block transfer of electrons to ubiquinone

2. Antimycin A (piscicide/furgicide)

   • Blocks transfer of electrons from ubiquinone to cytochrome c reductase

3. Cyanide (CN^-), azide (N_3^- molecule), carbon monoxide (CO)

   • Blocks transfer of electrons to O₂ (cytochrome oxidase) at the last step

ATP Synthase

• Highly conserved (bacteria, archaea, plants, animals, fungi, etc.)

• Fully reversible:

  • ATP Synthesis: ADP + P_i → ATP

  • ATP Hydrolysis: ATP → ADP + P_i

• 2 Domains:

  • F₀ = transmembrane domain; creates H⁺ channel

    • 3 H⁺ → 1 ATP

  • F₁ = “lollipop head” = catalytic domain (creates ATP molecule)

• Acts like a molecular “turbine”: flow of H⁺ through F₀ generates rotational energy

• Produces ~100 ATP/second

Drugs that Block ATP Synthase

1. Oligomycin

   • Antibiotic-produced Streptomyces bacteria

   • Poisons ATP synthase and blocks F₀ H⁺ ion channel

     • Prevents chemiosmosis from occurring

     • Can still make ATP by glycolysis

       • However, builds up too much lactic acid and acidifies blood pH and urine (fatal)

2. DNP = dinitrophenol

   • Affects ATP synthase indirectly by inserting itself into the inner mitochondrial membrane and acts as a H⁺ channel

     • Dissipating H⁺ gradient, preventing ATP synthase from tapping into a pool of energy that would have been made if all the H⁺ was transported across the membrane/H⁺ bypasses the ATP synthase; more heat is generated

     • Metabolic rate will increase in oder to compensate for the less amount of ATP being made (more fatty acid oxidation)

   • Very easy to overdose on it because if used as a weight loss drug, the therapeutic dose is very close to the toxic dose (now banned in U.S.)

Chloroplast vs. Mitochondria

Light reactions of photosynthesis by chemiosmosis, using an ETC, H⁺ gradient, and ATP synthases.

• Chloroplast has more compact spece

• Mitochondria permeable to water

• Chloroplast

  • 2 pH unit difference

  • 100x more H⁺ in thylakoid lumen vs. stroma