Chapter 11

* Principles of Membrane Transport * Given enough time, nearly all molecules will diffuse across a membrane * Smaller, hydrophobic/non polar molecules diffuse rapidly * Larger molecules, and charged ions move much slower * So mechanisms are needed * 2 Main types of Membrane Transport Proteins * Transporters * Bind to a specific solute and undergo shape change to move solute through membrane * Channels * Much more weakly interact with molecules * Create pores that allow specific molecules to pass through * Allows much more rapid transport * Passive/Facilitated Transport * Used by all channels and some transporters * This uses no energy and moves molecules “downhill,” with their electrochemical gradient * Active Transport * Used by transporters, here usually called pumps * Requires energy, ATP * Move molecules against the electrochemical gradient * Transporters and Passive Transport * Glucose Transport * Passive transport * Cooperative transport coupled with the transport of Na+ * Binds 2 Na+ and 1 glucose * The binding of either ligand, glucose or Na+, increases binding of the other. The concentration of Na+ outside the cell is greater, so the net movement transports more of both ligands into the cell. * Transporters and Active Transport * 3 Main Ways of active transport * Coupled Transport * Couple the uphill transport of one molecule to the downhill transport of another * ATP Driven * Couple uphill transport to hydrolysis of ATP * Light Driven * Couple uphill transport with energy from light * Mainly in bacteria and archae * Secondary Active Transport * Ion driven transporters * Uses gradient of one to transport the other against its gradient * Primary Active Transport * ATP driven transporters * 3 Types of Transporter-Mediated transport * Call all be used for both active and passive transport * Uniports * Mediate transport of one molecule at a time * Symport * Coupled simultaneous transport in one direction * Antiport * Coupled transport in opposite directions * Na+ Glucose transporter is a coupled symport * Lactose Transport * Lactose permease is the transport protein * Coupled symport * Has 12 alpha helices that slide and move around, occasionally opening a gap with H+ and Lactose binding sites, then exposing those sites to the inside of the membrane * Transcellular Transport * Transporters are sometimes non-uniformly distributed to provide a net movement of solute in one direction * Glucose from the intestines into the blood * 3 Types of ATP Driven Pumps * P-Type * Phosphorylate ATP to give energy to pump ions * F-Type * Multi-subunit turbine like protein * Utilizes H+ gradient to synthesize ATP from ADP * Not in plasma membrane, but in organelle membranes * V-Type * Similar, but only pump H+ to acidify compartments like lysosomes. Do not create ATP * ABC Transporter * Use ATP to transport small molecules. Not ions, like P and F Type. * Ca 2+ Pump – P-type * Typically low concentration of Ca 2+ inside cells * P-type pump on SR membrane of skeletal muscle cells * Pumps Ca 2+ from the cytosol into the SR lumen * Binds Ca 2+ and ATP – calcium first * Phosphoylates ATP * Moves phosphate group to Aspartic Acid * Causes shape change, possibly rotating activator domain 90 degrees, releasing Ca 2+ in the lumen.

* Na+ - K+ Pump * Typically high concentration of k+ in cells * Low concentration of Na+ in cells * ATP driven Antiport P-type * Actively pumps Na+ outside of cells, and K+ into cells, both against gradient. * Can be experimentally driven in reverse, and will synthesize ATP from ADP * Used to maintain osmotic balance and in turn volume of cell * ABC Transporter * TMD * Transmembrane domain * NBD * Nucleotide binding domain * Atp binding domain * Prokaryotes * Can move solutes either way, usually into a cell * First Binds small molecule * Then ATP binds, changing shape and exposes molecule binding to other side of membrane * ATP hydrolyzes, releasing molecule to other side of membrane, and re conforms to original shape * Eukaryotes * Basically the same thing, but usually pumps solute out of the cytosol, or into a membrane compartment. * Osmotic Balance * There are many solutes inside of a cell * This would normally cause water to continuously move into the cell, possible causing it to lyse. * To counteract this… * Animals * Actively pump out ions, maintaining a gradient that does not allow in enough water to cause problems * Plants * Have more rigid cell walls, that wont expand as much, and build up turgor pressure inside the cell that, at equilibrium, will force out as much water as is taken in * Double membrane Transport * Solutes diffuse through channel in outer membrane * Then bind to periplasmic substrate binding protein in the periplasmic space * This binding shape changes the protein, allowing it to bind to an ABC transporter in the inner membrane * The solute is transferred to the ABC transporter, and then actively diffused through ATP hydrolysis into the cytosol * Ion Channels * Transport ions Passively * The ions are let in individually, and each channel has specific ions it allows in * Distinguishes them by size, only certain ones fit through the narrow Selectivity Filter * Not continuously open, but change from open to close * Gated Channels * Voltage Gated Ion Channels * Mechanically Gated Ion Channels * Ligand Gated Ion Channels * Voltage Gated Ion Channels * Neurons, muscle, endocrine, and egg cells contain these channels * Generate the action potential * First by depolarizing the membrane * Shift to less negative charge inside the cell * Signals cause the opening of Voltage Gated Na+ channels * Allows small amounts of Na+ inside cell down its gradient * This influx causes positive feedback which opens more voltage gated Na+ channels * At a point, the channels automatically inactivate themselves * This forces the action potential in one direction * Then K+ Voltage Gated Channels open * This releases K+ ions, bringing the cell back to resting potential * Transmitter Gated Ion Channels * Located on the post-synaptic neuron in the synaptic cleft * Leave the pre-synaptic cell in vesicles * Channels on the post-synaptic cell bind a neurotransmitter and open transiently, briefly changing the permeability of the membrane * Unless it stays open for a long period of time, this will not always cause an action potential * Excitatory Neurotransmitters * Open cation channels, causing an influx of K+, moving the membrane to the threshold of action potential * Acetylcholine, glutamate, and seratonin * Inhibitory Neurotransmitters * Open either Cl- or K+ channels * This suppresses excitatory transmitters from changing the membrane potential * GABA and Glycine * Neurons * Myelin sheath greatly increases propagation of signals * Multiple sclerosis people don’t have myelin sheath and are fucked * Salutatory conduction * Faster by jumping gaps, and more energy efficient * Nodes of Ranvier * Gaps in the sheath * Where most Na+ channels are found * Acetylcholine Receptor * Acetylcholine is released from neuromuscular junction * The synapse of a motor neuron and a skeletal muscle cell * Pentamer * 5 trans membrane protein complex * 2 acetylcholine’s bind, transiently opening the channel * they are soon hydrolyzed by an enzyme, and the channel closes * if it remains open too long, it will inactivate itself * when open, moved Na+, K+, and Ca+ through, according to their concentration gradients, with little selectivity * Mainly allows large influx of Na+, causing depolarization of membrane that signals muscle cell to contract * Process of Muscle Cell Stimulation * Nerve impulse reaches nerve terminal, depolarizing terminal plasma membrane. * This transiently opens voltage gated Ca+ channels, which at high concentration outside the cell, flood inside the cell * This signal causes release of acetylcholine in synapse * Acetylcholine binds to its receptors on the post-synaptic cell * This opens the cation channels, allowing Na+ to enter the post-synapstic cell * Causes membrane depolarization * Depolarization opens more Na+ voltage gated ion channels, allowing in more Na+. Triggers action potential down entire cell * Depolarization opens voltage gated Ca2+ channels * This causes Ca2+ channels in the sarcoplasmic reticulum to open as well, flooding the cell with Ca2+, which is what triggers the muscle cell to contract

Chapter 12

Organelles and Transport * Topologically equivalent compartments * Nucleus and Cytosol * ER, Golgi apparatus, endosomes, lysosomes, transport vesicles, and possibly peroxisomes * Mitochondria * Plastids – Plants only * Protein movement between compartments * Gated Transport * Proteins moving between cytosol and nucleus through nuclear pores * Specific active transport, and diffusion of small molecules * Transmembrane transport * Moving proteins from cytosol to topologically distinct area * Uses Protein Translocators * Protein sometimes must be unfolded and snaked through membrane * Vesicular Transport * Use vesicles and must go to topological equivalent area * Sorting Sequences * Signal Sequence * Amino acid sequences found on the N-terminus of the protein * Signal Patch * Multiple sequences in the middle of the protein that come together folding the protein * Specifies destination * Those going to the ER usually have a N Terminus sequence of 5-10 hydrophobic amino acids * Many will go from there to the Golgi * But a 4 amino acids sequence on the C Terminus stay in the ER * Those going to Mitochondria have alternating positive and hydrophobic AA sequences * Those going to Peroxisomes have 3 AA sequence on C Terminus * Complementary Sorting Receptors * Recognize these sequences and take them where they need to go * Signal Peptidases * Usually remove these signal sequences * Nuclear Envelope * Inner Membrane * Anchor site proteins to attach chromatin and Nuclear Lamina * A protein meshwork that stabilizes the nucleus * Outer Membrane * Continuous with the ER * Rough with ribosomes * Proteins made on this membrane are moved to the perinuclear space between the membranes, which is continuous with the ER lumen * Nuclear Pore Complexes * 30 NPC proteins * Nuclear Localization Signals * Signals for the import into the nucleus through an NPC

* Nuclear import Receptors * Recognize specific molecules with nuclear localization signals * Soluble cytosolic proteins bind both to nuclear localization signal and NPC proteins * They find FG-repeats (Phenylalanine and glycine) on fibrils and on internal proteins and continuously bind and dissociate them to make their way through the pore. * Once inside, the receptor dissociates from the protein and leaves * Some use adaptors * Adaptor binds to nuclear localization signal on protein, and import receptor binds to localization signal on adaptor * Export works the same but backwards * Nuclear export receptors bind to nuclear export signals on molecules to be exported and then bind to NPCs * Both receptors belong to family of Nuclear Transport Receptors * Karyopherins * The cell gets the energy for this through the hydrolysis of GTP * By the monomeric GTPase Ran * Ran is found in and out of the nucleus, and is required for both import and export * Ran exists as GTP or GDP * GAP triggers GTP hydrolysis : Ran-GTP to Ran-GDP * GEF triggers Ran-GDP to Ran-GTP * Ran-GAP is in the cytosol * Ran-GEF is in the nucleus * So the cytosol contains manly Ran-GDP * Nucleus contains mainly Ran-GTP

* Nuclear Lamina * Nuclear side of the inner membrane * Meshwork of interconnected protein subunits called nuclear lamins * Lamins are phosphorylated * Breaks up the nuclear envelope for mitosis * Protein Translocation to mitochondria * Proteins destined for the mitochondria are made in the cytosol * Called mitochondrial precursor proteins * Moved post-translation to mitochondria * Sequence signals to move protein to mitochondrion space * Form amphiphilic a-helixes with charged residues on one side and uncharged on the other * Receptor proteins recognize this arrangement, not the actual sequence * Protein translocators mediate transport across membrane * TOM complex * In outer membrane * Transfers through outer membrane * All mitochondrion proteins go through here first * TIM complexes * In inner membrane * Transport inner membrane * TIM 23 * Helps move some proteins into inner space, and helps get transmembrane proteins into the inner membrane * Has hydrophobic a-helix that spans into the outer membrane * TIM 22 * Helps insert a subclass of inner membrane proteins * And multipass proteins * Including ATP, ADP, and phosphate * SAM complex * In outer membrane * Helps fold and position B-Barrels in outer membrane * Very similar to a membrane protein in bacteria, further proof of endosymbiosis * OXA complex * In inner membrane * Transports proteins synthesized in the mitochondria to the inner membrane * Requires membrane potential on inner membrane * Negative on the inner inside, positive on intermembrane space * Chaperone proteins from Hsp 70 family keep precursor proteins unfolded * ATP hydrolysis required to remove chaperones * Unless the protein is experimentally left unfolded * Mitochondrial Hsp70 chaperones pull the protein into the inner space after getting through TIM * ATP required to again release the chaperone * Mitochondial Hsp60 sometimes used to refold the protein once inside the mitochondria * Beta Barrels are snaked through TOM to the intermembrane space * Chaperones keep it from folding * It then goes to SAM, which puts it in the outer membrane and helps it fold * Several routes to inner membrane * Goes through TOM and only signal sequence goes through TIM23, the signal sequence is followed by hydrophobic sequence that stops the transfer. TOM pulls the rest into the intermembrane space, the signal sequence is cleaved inside the inner space, and the hydrophobic is released by TIM23 and left in the inner membrane with its hydrophobic residues. * Same as the first, but then TIM23 pulls it all the way into the mitochon space. Signal sequence is cleaved. Second hydrophobic sequence right behind it. That binds to OXA complex, which puts it in the inner membrane. * This does the same as either of those, but then its hydrophobic anchor to the inner membrane is cleaved and it stays in the intermembrane space * Multipass proteins are snaked through TOM, left in the intermembrane space where chaperones guide them to TIM22, which inserts them, using membrane potential, into the inner membrane

* Transport into Chloroplast * Like mitochon, except requires ATP and GTP hydrolyses * Precursor protein has 2 signal sequences, a chloroplast followed by a thylakoid sequence * Just like mitochon, but has to go through one more membrane to the thylakoid space * Goes through first two membranes * Once inside, the first signal sequence is cleaved * Then has 4 routes to get into thylakoid * Sec Pathway * Requires ATP and electrochemical gradient * SRP Pathway * Requires ATP and electrochemical gradient * TAT Pathway * Requires H+ gradient * Spontaneous insertion * Requires nothing * Transport into Peroxisomes * All cells have have peroxisomes * Have no of their own DNA, must import all proteins * Contain many oxidative enzymes * Believed to be an old organelle, before mitochondria, that dealt with oxygen without creating ATP. Then mithochon came in and did similar things, but created ATP. * also catalyze first reaction in creating plasmalogen * main phospholipid in myelin * Glyoxysomes * In plants, peroxisomes that use the glyoxylate cycle to breakdown fats to sugars * Import * Short AA signal on C-Terminus (Ser, Lys, Leu) * Some near the N-Terminus * Peroxins * 23 distinct proteins involved in import * powered by ATP hydrolysis * Pex5 recognizes sequence, takes to pex14 membrane protein, which inserts protein into lumen. Pex5 dissociates.

* ER * ER membrane is site of production of all transmembrane proteins and lipids * Import is done co-translationally * Smooth ER is for budding off to go to golgi * Import * All contain ER signal sequence * Some destined for ER membrane, lumen, or membrane and lumen of other organelles, or out of the cell * SRP * Signal recognition particle recognizes and binds to signal sequence * 6 polypeptide chains around a small RNA molecule * SRP Receptor * In the ER membrane * 2 subunits, an integral beta unit and a peripheral alpha unit * SRP binds to large subunit of ribosome, binding the ER signal sequence on the protein as it emerges, and stops elongation factors from binding, and causing full translation of the protein in the cytosol before getting to the ER * SRP-Ribosome complex then binds to SRP receptor, and integral ER membrane protein * This receptor brings the complex to a translocator that transmits the protein through the membrane * The translocator is a aqueous pore * Contains the Sec61 complex * 3 subunits * alpha helixes black the pore * open only transiently to allow insertion, and keep ions like Ca2+ inside * pore can also be opened sideways to allow membrane proteins to be inserted * Requires no extra energy, being pushed through by translation * To import post-translation * Prokaryotes * Accessory proteins are needed by the translocator to put protein through membrane * SecA protein is on the cytosolic side, and with each hydrolysis of ATP, pushes a chunk of the protein through * Eukaryotes * BiP * Binding protein in ER lumen that binds and unbinds through ATP hydrolysis, slowly pulls protein through

* Insertion of single pass transmembrane proteins * The ER signal sequence is recognized twice * Once by the SRP, and once at the translocator to open the pore * Operates as a start-transfer signal * During translation, signal sequence is bound to Sec61 and the hydrophobic lipid membrane. * After protein is translated, the signal sequence is cleaved by signal peptidase * The translocator then has to open laterally to allow the protein inside to integrate into the membrane * 3 methods to integrate a single pass membrane protein * ER signal sequence on N-terminus initiates translocation, and a later hydrophobic sequence acts as a stop-transfer sequence. From here, the translocator opens laterally, integrating the hydrophobic sequence into the membrane * ER Signal sequence is in middle of protein. SRP binds the internal sequence and brings the SRP-Ribosome complex to the translocator. The protein is put through the membrane till gets to the hydrophobic signal sequence, which remains as the single pass part of the protein. * If positive AAs precede the signal sequence, the C-Terminus will be in the lumen * If positive AAs follow the signal sequence, the N-Terminus inserts into the lumen * Insertion of multi pass transmembrane proteins * The SRP scans N-C terminals as protein is synthesized for hydrophobic sequences. The first it finds is the start-transfer sequence. This sets the reading frame, as the next will now be a stop sequence, even though both are similar to each other. * ER proteins * Some are meant to stay there * ER retention signal * 4 AAs at C-Terminus * Glycosylation * An precursor oligosaccharide of 14 sugars is attached to Dolichol, a lipid * Attached by phosphate bond that gives energy to drive next reaction * Oligosaccharyl transferase enzyme transfers the oligosaccharide to the protein * One of these enzymes for every transloctor * Most glycosylations are N-Linked, and the sugars are bonded to Asparagine * Some are O-linked, bind to hydroxyl groups * Calnexin and Calreticulin * Chapernones – lectins * Bind to oligos on incompletely folded proteins * Unfolded proteins have several glucoses at the end of their oligosaccharides * These are all trimmed but one * Calnexin binds this final glucose * Glucosidase removes the last glucose releasing it from calnexin * Glucosyl transferase determines if the protein is full folded or not * If not, it adds another glucose to it, which send it back to calnexin * Once it is finally fully folded, can exit the ER wherever it needs to go * Misfolded proteins are bound by chaperones and retrotranslocated out of the ER lumen * N-glycanase removed the oligo * ER bound ubiquitin conjugating enzymes add ubiquitin to the misfolded proteins, that end up in a proteasome and are degraded * Unfolded Protein Response * Accumulation of unfolded proteins triggers unfolded protein response * Increases transcription of ER chaperones and proteins involved in retrotranslocation and cytosolic degradation * How is this response sent to the Nucleus? *