BIO 266 Midterm 2 Notes PDF

lOMoARcPSD|47133420 BIO 266 Midterm 2 - Info Material Cell Biology (Concordia University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Elisabetta Ferro ([email protected]) ...

lOMoARcPSD|47133420 BIO 266 Midterm 2 - Info Material Cell Biology (Concordia University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 BIO 266 Midterm 2 Unit 4 - Membranes need to be uid in order for the proteins to move (change shape to let ions) - Membrane proteins  very important o Lipids  fundamental structure of a membrane o Proteins  carry out specic membrane funcons - Types of proteins  3 o (1) integral proteins  imbed into the membrane  don’t need to completely pass through  can pass through the membrane more than one me  Has an amino terminus and a carboxy terminus  where it starts and where it stops  Monotopic protein (amphipathic protein)  imbedded in membrane, does not pass it  “in-out” protein if it only spans the bilayer once  Hydrophobic region (uncharged + non-polar amino acids)  middle  anchors the protein to the bilayer through Van der Wal interacons  Hydrophilic parts (charged, polar amino acids)  ends of the protein  outside the bilayer  Odd numbered membrane-spanning domains  “in-out” orientaon  one end is on either side of the bilayer  Even number of membrane spanning domains  2 possible orientaons  “in-in”  same side of the bilayer  “out-out”  same side of the bilayer o (2) Lipid-anchored proteins  Protein does not embed in membrane  anchored to the membrane covalently through a lipid of some sort  3 types:  GPI-anchored o Anchored to a glycosylated phosphadylinositol o Protein needs no specic hydrophobic/hydrophilic parts since it’s not interacng with the membrane o Found in externa side of the plasma membrane  Fay Acid anchored o Protein linked to a fay acid  Isoprenylaon anchor o Protein can be aached to mulple isoprene (5 carbon groups) o Common on small GTPases o (3) Peripheral proteins Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Associates with some other protein that is in the membrane through weak electrostac bonds (easily soluble)  Enrely located on the outside of the bilayer  extracellular side or cytoplasmic side  Associates with membrane through covalent bonds  Some associate with lipids due to changes on the amino acid  associate with polar head groups  Can be stripped o membrane easily with a high pH or high salt concentraon  Associaon with membranes can be dynamic and dictated by condioned inside the cell - Funcons of membrane proteins o (1) transport for charged ions (H+ gradient in the mitochondria and lysosome) (and or) large molecules (sugars, nucleodes and amino acids) o (2) link proteins on  Cytosolic face of the membrane to proteins on the noncytosolic face  integrins link the acn cytoskeleton to the extracellular matrix protein  Anchor proteins on either side of the membrane o (3) receptors  detect chemical signals on the face of the membrane and relay them to the other face  hormones o (4) enzymes catalyze rxns on either side of the membrane - Proteins cross the membrane as an alpha helix o Between 2 amino acids  pepde bond between the carboxyl and the amine o Oxygen pulls more electrons towards itself than Carbon o Nitrogen pulls the electrons towards them towards itself than Hydrogen  Result  Oxygens are slightly negave, Hydrogen atoms are slightly posive therefore they form hydrogen bonds o H forms a hydrogen bond with an Oxygen that is 4 amino acid residues away  forms alpha helix structure (a single polypepde chain that turns around itself to form a rigid, spiral-like structure)  To count residues, count the r-groups o Proline  never found in an alpha helix since it’s conguraon of its r-group o Number of alpha helixes that span the bilayer  16.7 o Single alpha-helix that passes the membrane once  needs have (only) hydrophobic, non-polar r-groups  Can have charged r-groups in mulpass proteins  r-groups interact with one another  interact with a part of the surrounding lipids  found on proteins that act as receptors for extracellular signals  extracellular part of protein binds to the signal molecule, the cytosolic side signals to the cell interior o amphipathic alpha helices Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  alpha helices that fold a certain way where the face contains hydrophobic nonpolar amino acids and the opposite face contains charged or polar amino acids  found in mulpass proteins  form a hydrophilic pore (job of hydrophilic amino acids)  funcon of hydrophilic pore  transport large molecules and small charged molecules across the membrane  hydrophobic amino acids interact with the bilayer - Proteins can also cross the membrane as beta-sheets/barrels o Form hydrogen bonds with residues that are far-removed o Strands can run parallel or anparallel o Has a kinked structure o Stabilizaon of strands  hydrogens bonds with neighboring strands  forms beta-sheet o Beta-sheets can curve around to form a beta-barrel o Hydrophobic poron of beta-barrel  interacts with lipids in the bilayer o Hydrophilic poron  inner poron of the barrel that line the aqueous pore o Pore  transports large molecules and small charged molecules across the membrane - Extract a protein from the phospholipid bilayer o Treatment that disrupts the bilayer  disrupt hydrophobic interacons that take place between the lipid tails and hydrophobic residues (Van der Wal interacons) o Common reagents  detergents (small, amphipathic, lipid like molecules)  SDS  strong ionic detergent  denatures protein  Triton X-100  mild non-ionic detergent does not denature protein o When the detergents are mixed in great excess with the lipid bilayer  Hydrophobic ends binds to the hydrophobic region of the transmembrane protein + hydrophobic tails of the bilayer Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  With water  arranges the hydrophilic heads which brings the protein structure into soluon as a protein-detergent complex - Detergents vs membrane lipids o Detergents:  have single hydrophobic tail  Shaped more like cones  Cluster into micelles o Lipids:  Has 2 hydrophobic tails  Shaped more like a cylinder  Cluster into a bilayer - Restricon of the movement in the membrane proteins  forms membrane domains o Relies on protein-protein interacons  (1) between similar proteins cause aggregaon (go to the same area)  Cluster of bacteriorhodopsin molecules on the surface of a Halobacterium halonium cell  (2) between membrane protein and an extracellular protein  CAMs bind between the cell and the extracellular matrix  (3) between membrane protein and the underlying, intramolecular cytoskeleton  Membrane proteins of the erythrocyte are held in place by anchoring to the cytoskeleton  (4) between membrane proteins on the surface of two cells  CAMs bind between cells  can be homophilic (bind to the same molecule on a dierent cell) or heterophilic (bind to a dierent molecule on a dierent cell) Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Immobilizaon of membrane proteins accounts for their dierences in diusion constants within the membrane  uidity of the membrane inuences the mobility of the membrane - Localizing membrane proteins  lipid ras o Domain on the membrane that has a high concentraon of specic lipids  In plasma membrane  enriched in cholesterol, GPI-anchored proteins glycosphingolipids with long saturated hydrocarbon tails o Rigid and thicker than the surrounding membrane Unit 5: Membrane Transport - Arcial membranes  not equally permeable to all substances o Smaller molecules  more permeable  more likely it is to cross the membrane Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Impermeable to large uncharged polar molecules + small charged molecules o Permeable to large uncharged polar molecules and charged molecules o Transfer of water soluble proteins depends on membrane transport proteins  Transport proteins  specic to a type of molecule  specicity  Builds up uneven concentraon on either side of the membrane  direconality o Each membrane has its own characterisc set of transport proteins  Depends on their funcon and what the organelle needs - Ion concentraon gradient  in result of specic transporters that move specic ions o If unbalanced, cell could be torn apart o Outside the cell: posive charge  Na+ balanced by negave charges  Cl- o Inside the cell: posive charge  K+, negave charge  nucleic acids and proteins o Surface of the plasma membrane  posive and negave charges accumulate  membrane potenal  Inside the cell  negave, outside the cell  posive Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - How substances pass through membrane barriers o Types of transport  (1) passive transport  needs no external energy, use concentraon gradient to move molecules from one side to another (high concentraon to low concentraon)  (2) acve transport  moves molecules against their concentraon gradient and therefore, requires external energy - Passive transport  diusion o Gases (oxygen, carbon dioxide) , hydrophobic molecules (benzene) and small polar uncharged molecules (water, ethanol)  All dissolve in the lipid bilayer, diuse across it and dissolve in the aqueous soluon  Rate of diusion:  All have a paron coecient (measure of a molecules ability to paron between aqueous and hydrophobic environments)  Size  smaller moves faster than larger  If 2 molecules have equal paron coecient then the smaller one diuses faster than the larger one  Direcon of transport  Moves with the concentraon gradient  molecule moves in either direcon unl an equilibrium is reached - Facilitated Diusion o Passage of polar and charged molecules is mediated by proteins to bring them across a membrane  Proteins are called carriers or channel o Requires no energy since the protein moves with the concentraon gradient  therefore has no direconality o Large uncharged molecules (amino acids, nucleodes, sugars) and charged molecules cannot dissolve in the lipid bilayer  Cannot dissolve in the bilayer - Acve transport Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Moves molecules against their concentraon gradient therefore requires energy (hydrolysis of ATP) o Molecules move in one direcon  Na+/K+ pump, ATPase in the lysosome - Class of transport proteins o (1) ATP-powered pumps 1-1000 molecules/sec  Couples ATP hydrolysis to transport molecule against their [] gradient o (2) Channel proteins (mostly for ions) 10^7-10^8 molecules/sec  Does not directly interact with the molecule  just opens and closes  allows a large ow of molecules to pass through  Goes with the concentraon gradient o (3) Carrier proteins (transporters)  !0^2-10^4 molecules/sec  Physically interacts with the substance by binding water-soluble molecules on one side of the membrane and deliver them to the other  Need a conrmaon change  Can only bind a certain number of proteins at a me - Subdivision of channels and carriers o Uniporter  Selected for one type of protein  moves one thing down it’s gradient (ex: sodium channel  facilitated diusion) o Symports  both molecules move in the same direcon o Anporter  2 molecules move in opposite direcon  one goes with their concentraon gradient while the other goes against it - Uniporter vs Simple diusion Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (1) rate of diusion  Higher rate in uniporters than with simple diusion o (2) Paron co-ecient  Irrelevant for uniporters  no contact with the hydrophobic lipid environment o (3) Uniporter transporter has a limit  Limited by the number of uniporters on the membrane  Diusion is not limited in such manner o (4) Transport with a uniporter  More specic, diusion lets any* molecule through o Km  anity for an enzyme for its substrate  The lower the Km  the ghter the binding between the substrate and the enzyme  higher anity  Ex: GLUT1 has a Km of 1.5mM for glucose and 30mM for galactose  therefore, even if there is a low concentraon of glucose (compared to galactose), glucose will sll bind to GLUT1 over galactose - Secondary Acve Transport o Anporter + symporter use electrochemical gradient to move one molecule against its concentraon and the other with its concentraon gradient o Binding of the two dierent molecules is cooperave  conrmaon change necessary to deliver the two molecules to the other side of the membrane only happens when both molecules are bound to the transporter - Sodium/glucose symporter eecveness o Using sodium electrochemical gradient generated by sodium potassium pump  can make internal glucose concentraon 30 000X greater than the external concentraon Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Classes of pump structures o Pump: something that uses ATP in order to move something against its concentraon gradient o P-class: herterotetramer with 2 subunits (alpha and beta component)  Beta component  phosphorylated during transport  Sodium/potassium pump, ATPase o V-class: mulple subunits, transports protons against their gradient  Acidies lysosome and vacuole o F-class: mulple subunits, related to V-class, generates ATP, pumps only protons  Inner membrane of mitochondria, thylakoid and bacterial plasma membrane o ABC superfamily: uses ATP to open channel  allows molecules to move with the concentraon gradient  Transports sugars, amino acids, phospholipids, proteins Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Sodium/potassium ATPase o E1 conrmaon: 3 high anity sodium binding sites, 2 low anity potassium binding sites  cytosolic-facing surface o 3 sodium bind to bind to the protein due to the high anity  despite low intracellular sodium concentraon  Potassium cannot bind to the receptors  low anity despite high concentraon o ATP binds to its site on the cytoplasmic site  ATP is hydrolyzed to ADP  phosphate is transferred to a specic asparc residue  forms a high-energy acyl phosphate bond o Change in conrmaon (E2)  from E1 to E2, the 3 sodium ions become accessible to the exterior face  E2 conrmaon  3 low anity for sodium, 2 high anity for potassium  Sodium ions dissociates and potassium associate with their high anity sites o Aspartyl-phosphate bond in E2 is hydrolyzed  E2 to E1 conrmaon  2 bound potassium ions become accessible to the cytosolic face  Due to the low anity sites, potassium is released in the cytosol o Restart - Calcium ATPase  muscle cells (contracon), nerve cells (neurotransmier release), ferlized zygote (start development), liver cells (glycogen breakdown) o E1 conrmaon  has a high anity for calcium o Phosphorylaon of ATP  conformaonal change into E2  exposes calcium to the cell exterior o Dephosphorylaon  conrmaon change into E1 - ABC transporters o 2 domains  transmembrane domain (6 alpha-helices per monomer  12) + nucleode binding site o CFTR transport channel  ion channel that needs energy to open  not a pump even if it looks like one o First one discovered in humans  MDR (muldrug resistance) - Membrane transporters in Health and disease Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Potassium/Proton ATPase  makes an environment acidic in the stomach  Drugs: Prilosec, Nexium, Prevacid  Acidity can also be neutralized by basic anions o Sodium/potassium ATPase  inhibited by ouabain (alters sodium gradient  aects sodium/calcium exchanger  calcium stays inside the cell instead of going outside) o CFTR  ABC family member  Chlorine transporter coupled with ATP hydrolysis and binding  Hydrates mucus layer in the lungs so that bacteria won’t grow on it  if CFTR is nonfunconal, mucus layer thickens and bacteria acclimates which causes infecons o Common mutaons  deltaF508  F deleted at protein 508  causes protein to not fold properly at body temperature but sll funconal + folded at low temperatures  G551D  glycine is replaced by asparc acid  Channel does not open properly  Kalydeco xes it by opening the channel more - Ion Channels o (1) selecvity some ions pass through while others don’t  Depends on diameter and shape  ion  Charges that line the inside of the ion channel o (2) gang  Channels are not connuously open  open in response to a smulus for a short period in me  Does not undergo a conformaonal change  Types of gang (3)  (1) Voltage-gated  responds to changes in electric potenals across the membrane o Found in nerve cells, muscles cells, egg cells and plants o Opening channel  alters the membrane potenal (again)  can acvate or disacvate other voltage gated channels  (2) Ligand-gated  (neurotransmiers) response when a ligand is bonded to a receptor  (3) Mechanically-gated  response to a mechanical force (auditory hair cells use this) o Sound waves cause channels to open  ions ow in  aects voltage gated channels  signal goes through the brain Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Nerve Impulses  use of ion channels o Have a membrane potenal of (approx.) -60mV  cell is slightly negave o Signal (from another neuron) causes depolarizaon  opening of voltage-gated sodium channels at -40mV  More channels open unl the membrane potenal is at +40mV o At +40mV, electrochemical force for sodium is 0  channels assume inacve state (closed enough where ions cannot pass but not sensive to polarizaon to the membrane  causes direconality)  ensures that signal will travel to the following neuron  causes voltage gated potassium channels to open  causes hyperpolarizaon (repolarizaon) to reinstall the membrane potenal o At the axon terminus  voltage gated calcium channels open  Inux of calcium  causes vesicles to fuse with the membrane  Release of neurotransmiers across the synapc cle  bind to metabotropic ion channel and neurotransmier binding = 2 dierent proteins) and ionotropic (ion channel + neurotransmier binding = same protein) receptors on the postsynapc cle Chapter 6: Part 1 - Protein sorng in organelles  they have a specic locaon and how do they get there - Protein synthesis in eukaryotes o Some proteins are encoded by mitochondrial + chloroplast DNA  Synthesized by ribosomes inside the mitochondria/chloroplast  Incorporated directly into compartments within the mitochondria o Most proteins  Encoded by nuclear DNA  Synthesized by ribosomes in the cytosol  Delivered to the organelle of desnaon from the cytosol Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Protein sorng  process by which newly-made proteins are directed to the correct locaon o Protein has a sorng signal on a single sequence  Can be between 3-60 connuous amino acids  Somemes removed once proteins arrive to their nal desnaon  Ex:**KNOW THEM**  Lumen of ER  KDEL  Lys-Asp-Glu-Leu-Coo-  Insures proper import of protein into peroxisomes  SKL  Ser- Lys-Leu-COO- - Signaling  necessary + sucient for protein sorng o There are programs that look for stretches that are similar in proteins in the same area (ex: ER lumen  end with KDEL) o Necessary:  Remove KDEL from a luminal ER protein and see if it ends up in the ER  If not, then KDEL is necessary for ER import o Sucient:  Does it need co-requirements (anything other than KDEL to enter the ER lumen)  Put KDEL on a protein not found in the ER and see if that protein shows up in the ER o If it does, then KDEL is sucient enough to let the protein enter the ER lumen - 3 Steps in protein sorng Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (1) recognion of the signal sequence of the protein by a shuling cytosolic receptor o (2) targeng to the outer surface of the organelle membrane o (3) import of that targeted protein into the membrane or transport of the protein across the membrane of an organelle o General problem: how to transport proteins access membranes that are nearly impermeable to hydrophilic molecules - 3 main mechanisms to import proteins into a membrane-enclosed organelle o (1) Transport through nuclear pores  Transports specic proteins  Protein remain folded during transport o (2) Transport across membranes  ER, mitochondria, chloroplasts, peroxisomes  Needs protein translocators (proteins that allow specic proteins into ER)  Proteins remain unfolded to cross membrane o (3) Transport by vesicles  From ER onwards and through endomembrane system  Transport vesicles pinch o from ER membrane, delivers cargo by fusing with another compartment  Proteins remain folded - Nuclear Import o Proteins enter through nuclear pore complex (NPC)  has no direconality, small water-soluble proteins can move through the pore by diusion o Parts of the nuclear membrane + pore  Nuclear basket: brils inside the nucleus converge at their distal ends to form a basket  funcon is not well understood  Membrane ring protein: anchors NPC to nuclear envelope  Channel nucleoporins: line the central pore, makes a mesh like nature of the NPC  Scaold nucleoporins: stabilizes the curvature and anchoring of the nuclear pore  Cytosolic brils: helps channel cargo to the NPC Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Nuclear Pore import o Need to get a protein into the nucleus:  If protein is small enough can diuse on its own  Others need an NLS (nuclear localizaon signal) within the protein  stretch that is basic in characterisc (lysine and arginine)  (1) Protein’s NSL associates with imporn alpha which associates with imporn beta  (2) guilds into nuclear pore complex  (3) imporn complex binds to RAN-GTP  binds to imporn beta  (4) due to this binding, the complex breaks apart  (5) exporn binds to imporn alpha  takes it out, imporn beta bound to GTP  therefore we need gap to dissociate the complex (GAP is in the cytosol)  once imporn-GTP leaves the nucleus, GAP comes and dissociates them and RAN-GDP enters the nucleus in its’ acve form (meaning GEF is in the nucleus and quickly converts RAN-GDP to RAN- GTP) Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o GAP in cytosol, GEF in nucleus  RAN gradient  Inside of the nucleus  high concentraon of high Ran-GTP  Outside the nucleus  high concentraon of RAN-GDP o Protein that needs an NLS  GEF since it needs to be in the nucleus to perform its funcon o Ensures direconality to nuclear transport - Mitochondrial import o Needs a N-terminal sorng sequence (1)  If it goes to the inner mitochondrial membrane then it will need another one (2) o Sequences that target the matrix  rich in hydrophobic, posively charged and hydroxylated residues (Ser, Thr) but not acidic  forms amphipathic helix o Steps:  (1) precursor protein stays unfolded by Hsc70  needs energy from ATP hydrolysis Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (2) matrix-targeng sequence interacts with outer mitochondrial membrane receptor called TOM20 or TOM22  (3) receptor transfers the protein to the general import pore of the outer membrane  TOM40  (4) if the inner mitochondrial membrane touches (or is close to ) the outer mitochondrial membrane, protein passes through the import pore of the inner membrane  TIM23 &TIM17  (5) Matrix Hsc70 binds to TIM44  ATP hydrolysis powers translocaon of the protein into the matrix  (6) matrix targeng signal sequence gets cleaved by a protease  (7) protein folds with the help of matrix chaperons Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Proton electrochemical gradient generates oxidave phosphorylaon  Required for protein import into mitochondria  Ensures that acve mitochondria are imporng proteins  Uncouplers block import o Targeng proteins in the intermembrane (mitochondria) space  needs a second hydrophobic targeng sequence  Does not allow protein to pass through TIM23/17 import pore Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Released into intermembrane  anchored intermembrane protease cuts the protein from the membrane  released into the intermembrane space - ER import o Most proteins that enter the ER begin to translocate across the ER membrane before the protein is completely synthesized  Ribosomes synthesizing the protein aach to the ER membrane o Co-translocaonal translaon - 2 types of ribosomes in the cytosol o (1) membrane bound  aached to the cytosolic surface of the ER  Synthesizing proteins that are translocated into the ER o (2) free ribosome  Unaached to any membrane and are synthesizing all of the other proteins - Steps to import a protein into the ER Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (1&2) emerging polypepde with the ribosome (with KDEL exposed) associates with SRP causes translaon to stop and delivers protein-ribosome complex to the ER o (3) SPR delivers ribosome/protein complex to SRP receptor  needs hydrolysis of GTP to do so o (4) ribosome/protein complex is transferred to the translocon opens  polypepde enters the tunnel in a loop  Hydrolysis of GTP by SRP and SRP receptor causes SRP to detach from the ribosome and SRP receptor from the translocon  recycled to do this to another ribosome-polypepde sequence o (5&6) translaon resumes and signal sequence is cleaved by a membrane bound protease signal pepdase  Connues synthesis and enters the lumen of the ER o (7&8) aer translaon is complete  ribosomes is released and protein is properly folded - Membrane proteins of the Plasma membrane. Golgi, lysosome and endosome o Inserted into ER membrane then transported to their correct locaon using sorng signals o 6 main types of membrane-anchored proteins (in  in cytosol, out  lumen or extracellular space)  (1) Type 1  single pass  cleavable signal sequence and a stop-transferase anchor (STA) sequence  acts as the membrane spanning domain o translocon opens to release this hydrophobic stretch into the membrane  uses SRP-SRP receptor to get to ER membrane  Nout-Cin Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (2) Type 2  Single pass  Non cleavable signal sequence  hydrophobic stretch appears and stops translaon  uses SRP-SRP receptor to get to ER membrane  Nin-Cout o Posive residues between amino end (N) and SA  cytosol  Use signal-anchor (SA) sequence  dual sequence (direcng protein to ER by the SRP)  (3) Type 3  Same as type 2 Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  BUT Nout-Cin  orientaon is due to translocon recognion of the hydrophobic stretch and posive residues that will make that part cytosolic o Posive residues between SA and the carboxy end (C)  cytosolic  Use signal-anchor (SA) sequence  dual sequence (direcng protein to ER by the SRP)  (4) Tail-anchored  Single pass  No cleavable sequence  Hydrophobic membrane-spanning sequence at C-terminus  Does not use SRP-SRP receptor o Uses GET1/2/3 system to get to ER  (1) protein fully synthesizes  fully expelled from ribosome  (2) GET3 (in ATP state) recognizes hydrophobic sequence at the C-terminus  binds to it and deliver it to GET 1/2 on ER membrane (receptor complex)  (3) ATP hydrolysis of GET3 releases protein in ER membrane  hydrophobic stretch imbeds in ER membrane  no luminal protein  (4) GET3 expels ADP and binds to ATP  recycled Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Posranslaonal inseron  Nin-Cout  (5) GPI-anchored  Enre protein is luminal (out)  Cleaved signal sequence at N-terminus  Uses SRP-SRP receptor to get to ER membrane o Embeds like a “type 1 protein”  uses STA sequence o Transamidase cleaves protein within ER lumen o Transamidase transfers it to assembled GPI anchor  Purpose of transferring one lipid anchor to another:  (1) GPI anchor more readily diuses in the membrane  (2) GPI can act as a targeng signal  Anchored at C-terminus to the membrane  transferred to GPI anchor Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (6) Type 4  Mulspanning  No cleavable sequence o Embeds into ER and determines orientaon  Uses SRP-SRP receptor for inseron of the rst membrane- spanning domain but not subsequent ones  Type 4-A  Nin-Cin  Type 4-B  Nout-Cin  Uses combinaons of stop-transfer anchor (STA) and Signal Anchor (SA) o If rst SA sequence is Type 2 SA where Nin-Cout  N is posive (in the cytosol only) o If the rst SA sequence is a Type 3 SA where Nout-Cin  C is posive (cytosol only) Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Hydropathic plots  helps determining the type of membrane protein o Looks at a sequence of 20 amino acids  gives it a score  If an amino acid is more hydrophobic  more posive the index  If an amino acid is more hydrophilic  more negave the index o Hydrophobic peak  cleavable sequence near Carboxy terminus  GPI protein - ER is the starng point for o (1) soluble proteins that will be secreted from the cell  hormones o (2) soluble proteins that are desned for the Golgi, lysosome or endosome  acid hydrolysis Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (3) membrane proteins that will embed in the Golgi, lysosome and endosomes or plasma membrane (sodium/potassium ATPase) - ER  ensures that proteins are properly modied, folded and assembled by quality control o 4 main modicaons  (1) Disulde bond formaon  (2) glycosylaon  adding and processing of carbs  (3) folding or polypepde chains and assembly of the mul-subunit complexes  (4) proteolyc cleavage of amino-terminal signal sequences - (1) Disulde Bond formaon o Proteins part of endomembrane system o Covalent bond formaon between thiol groups of cysteine residues either on the same protein (intermolecular) or on two dierent proteins (intramolecular) o Formaon of bond in the ER (only)  PDI (protein disulde isomerase)  Only secreted or luminal or extracellular domains of membrane proteins undergo this modicaon o Funcon: stabilize protein structure  Important for proteins that will be in extreme condions (low or high pH or high levels of proteases) - (2) Glycosylaon o Only glycosylates proteins with a sequence present  Asn-X-Ser or Asn-X-Thr  Where X is any protein o N-linked glycosylaon  oligosaccharide is added to amine group of asparagine  Needs ER membrane bound enzyme complex  oligosaccharyl transferase o Precursor oligosaccharide is transferred to the protein as the consensus sequence emerges from translocon  Assembled in a step-wise fashion on a lipid molecule o Trimming in the ER and Golgi usually does not involve these 5 sugar residues and they can be thought of as the core  Core  2x N-acetylglucosamine and 3x mannose Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Know carbs on glycosylaon  2x N-acetylglucosamine  9x mannose  3x glucose o Dolichol contains 75-95 carbons  sugars are added to it  Sugars are coupled with nucleode o (1) Assembly of 2x N-acetylglucosamine (GlcNAc) and 5x mannose  on cytosolic surface of the ER o (2) dolichol (with 7 sugar residues) ips using ippase into the lumen of the ER o (3) transporter moves residues to another dolichol phosphate o (4) remaining sugars are added one at a me unl the precursor is made Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Aachment of sugars to dolichol  UDP-GlcNAc  UDP-glucose  GDP-mannose o Inhibion of glycosylaon:  Tunicamycin  inhibits aachment of the rst GlcNAc residues to dolichol  proteins cannot get glycosylated  Increases unfolded protein response since proteins need to be glycosylated as a sign of protein folding o Roles of Glycosylaon  (1) promote folding of proteins  (2) stabilize proteins  (3) cell-cell adhesion on plasma membrane proteins  (4) act as a transport signal - (3) Folding o Molecular chaperones assist in protein folding by prevenng aggregaon of hydrophobic stretches o Two types of ER chaperones  Classical chaperones (Hsp70, Hsp90, GRP94)  Carbohydrate-binding chaperones (calnexin, calreculin)  Bind into polypepdes are monoglucosylated  Terminal glucose is removed and if folded the protein can exit the ER Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 Unit 6: Part 2 - Mechanisms that control exit of proteins from the ER include o (1) quality control  is the protein folded? Is the protein complex assembled? If not, it stays in the ER by chaperones o (2) Acve cargo selecon  specic cargo are collected in regions of the ER that will pinch o to form a transport vesicle  soluble cargo are recognized by membrane proteins that span the ER bilayer  membrane cargo recognized by cytosolic proteins that will aid in vesicle formaon - Some proteins belonging in the ER leave by accident o Proteins have targeng signal KDEL at the c-terminus interreacts with KDEL receptor o Receptor cycles between Golgi and ER  binds to KDEL proteins in the Golgi  releases them back into ER Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Resident ER proteins  have retrieval signal KKXX at C-terminus in cytosol o Recognized by COP1 coat (set of proteins that are needed to form transport vesicles from the Golgi to the ER) - Vesicle-mediate protein transport in conserved among eukaryotes  including yeast o Yeast temperature-sensive mutants are used to idenfy many of the proteins needed for this process and to understand the mechanism o Depending on the mutaon  inhibit a certain step in the endomembrane system - Formaon of transport vesicles o Driven by set of proteins that coat outside of newly formed vesicle  coat protein complexes o 3 classes of vesicles coats  (1) Clathrin  mediates transport vesicle formaon at the trans-Golgi (for transport to lysosomes (using endosomes) and at the plasma membrane (for transport to endosomes)  (2) COP1  mediates transport from cis-Golgi to ER and between dierent parts of the Golgi  (3) COP2 mediates transport from ER to the cis-Golgi - Funcons of protein coat on cytosolic surface of budding vesicles o (1) shapes the donor membrane into a bud o (2) helps capture cargo proteins into budding vesicles o Requires small GTP binding proteins called Rab proteins  GTP-acve and GDP- inacve form  GDP to GTP requires a GEF  acvates  GTP to GDP requires a GAP  inacvates Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Steps of COP2 coat formaon o (1) Rab protein (Sar1) is acvated by GEF  inserted itself into the membrane and starts curve it  can do so since it’s an amphipathic helix o (2) Sar1 recruits inner poron of COP2 coat made up of Sec23 and Sec24  Further bends the membrane  Sec24 acts as a cargo receptor for membrane proteins o (3) Sec23 and Sec24 recruits the outer layer of the COP2 coat (made up of Sec13 and Sec31) - Fusion of vesicle with target membrane o (1) Vesicle coat must be completely or mostly removed from the vesicle o (2) Vesicle must be specically recognized by the correct membrane o (3) Vesicle and target membrane fuse and mix to deliver contents from vesicle to target organelle - Vesicle Coat disassembly o Formaon of COP1 coat at Golgi needs  Arf1 (rab protein)  Cop1 complex o For COP1 and COP2 vesicles, uncoang requires  inacvaon of Rab proteins o Clatherin coat disassembly depends on lipid composion - Fusion of vesicles displays great specicity  proteins that disnguish each membrane in the cell  uses Rab proteins o Acvated form of Rab protein  bind to eector proteins Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Rab proteins on vesicle and target membrane can bind to eectors that contribute to vesicle tethering o 3 main steps in vesicle-mediated transport aer budding  (1) tethering  mediated by Rabs and their eectors, tethering factors and SNAREs  (2) Docking  mediated by SNARE pairing  (3) fusion  driven by SNARE zippering - (1) Tethering o Grabs vesicle, looks at it and if incorrect, sends it away o Inial contact between vesicle and target membrane o Occurs over a long distance o Classes of tethers  (1) Mulprotein complexes  up to 10 proteins, localize to disnct organelles  (2) Coiled-coil proteins  long alpha-helix that projects great distance from the target membrane o Each tethering factor is a Rab eector - Vesicle Docking o Stronger interacon between vesicle and the target membrane o Occurs on a sort distance o Mediated by SNARE proteins on vesicle (v-SNARE) and target membrane (t- SNARE)  All have a SNARE mof that allows it to interact with another SNARE protein  bundle of alpha-helices (4-helix bundle)  3 by t-SNARE, 1 by v-SNARE  trans-SNARE complex  Does not readily break apart  forms an energy favorable state Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Membrane fusion driven by SNARE pairing o Brings vesicle and target membrane into close proximity to displace water molecules surrounding the polar head groups of the outer leaet o 3 stages:  (1) Outer leaet mixing between the vesicle and target membrane  hemi fusion intermediate  (2) Expansion of hemi-fusion intermediate provides a surface for the inner leaet to fuse  (3) Fusion of inner leaets allows access of the soluble material in the vesicle and the target membrane to mix Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Vesicles-mediated transport reacons requires SANREs (v-SNARE and t-SNARE), Rabs + eectors  specic to each vesicle-mediated transport reacon o Requires factors that are common to each transport step:  (1) NSF  hexametric (6 copies of the same polypepde) ATPase  Aaches to cis SNARE complexes using accessory proteins called SNAP proteins  Hydrolysis of ATP breaks apart the stable cis SNARE complexes and allows SNAREs to be reused  (2) SNAP proteins - Protein arrives in the cis-Golgi  2 models to describe how it travels though the Golgi o (1) Vesicle transport model  Golgi cisternae are stac, stable compartments  Receive and transport cargo in antegrade direcon o (2) Cisternal maturaon model  Secretory cargo is stac and passively matures as Golgi enzymes from later compartments travel in retrograde-directed (trans-cis) vesicles  Golgi enzymes move, cargo is staonary - (1) Vesicle transport model o (1) Cargo is packed into vesicles that rst bud from the cis Golgi and fuse to the medial o (2) vesicles from the medial Golgi containing same cargo, bud then fuse with the trans o Cargo is physically transported in vesicles while the Golgi compartments never move Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - (2) Cisternal maturaon model o (1) Vesicles bud from each Golgi cisterna and contain Golgi enzymes specic to that cisterna o (2) Vesicles move backwards to an earlier Golgi Cisterna and deliver their contents there o (3) Overme cis Golgi acquires medial Golgi enzymes, converng it into medial Golgi enzymes o (4) At some me, medial Golgi shed its enzymes in vesicles and acquires trans Golgi enzymes o (5) Trans Golgi morphs into trans Golgi network (TGN)  vesicles will bud and fuse with the plasma membrane, endosomes or lysosomes o Cargo is never moved  its surroundings changed Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Golgi funcon  glycosylaon factory o Trans Golgi  adds galactose and other Carbs o Medial Golgi  Addion of GlcNAc, fucose and mannose trimming o Cis Golgi  mannose trimming - Unique modicaon  only in soluble lysosomal enzymes o Producon of mannose-6-phophate Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Delivery from TNG to lysosomes o Soluble lysosomal enzymes with mannose-6-phosphate (M6P) are recognized by M6P receptor  Only binds at a pH of 6.5-6.7  Releases at pH of an endosome (pH 6) o From the endosome, M6P receptor is recycled back to TGN o Phosphate is removed from the soluble enzyme which is then transported from the endosome to lysosome - Endocyc pathway moves material inside the cell o 2 main types of endocytosis  (1) bulk-phase endocytosis (pinocytosis)  non-selecve, can be clathrin- dependent or clatherin-independent Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (2) receptor-mediated endocytosis  selecve, clathrin-dependent, iniated by the binding of a ligand to its receptor o Clathrin forms outer layer of the coated vesicle  has a triskelion appearance o Adaptor proteins form inner layer of coated vesicles and engage the cytoplasmic tails of receptors  recruitment is facilitated by a lipid called phosphadyl inositol (4,5) bisphosphate  clathrin + adaptor proteins = “coated pit” - “coated-pit” evaginates while a small GTP binding protein called dynamin binds as a ring around the emerging stalk o Using GTP hydrolysis energy, breaks vesicle free from the plasma membrane o If non-hydrolysable GTP is formed, then the stalk connues to grow Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Uncoang the Clathrin  needs o (1) modicaon of the lipid that bind the adaptor proteins o (2) energy provided by the hydrolysis of ATP Hsc70 o Uncoated vesicles fuse to form early lysosomes - Endosomes undergo a “maturaon” process into a late lysosome o Late endosomes  lower pH than early lysosomes o Late endosomes  associate with a Rab7  early endosomes associate with Rab5 o Late endosomes  found near Golgi in the cell interior  Early endosomes are found near the plasma membrane o Late endosomes  round/oval structure  Early endosomes  complex structure - 3 fates for the receptor ligand complex o (1) low pH of early endosome causes disassociaon of the ligand from the receptor (LDL/LDL receptor)  Receptor is recycled and ligand is routed to the lysosome Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (2) ligand and receptor do not dissociate and the receptor shules ligand back to cell  transferrin/transferrin receptor) Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (3) ligand and receptor are both sent to the lysosome for degradaon (EGF/EGF receptor)  Receptor is tagged with ubiquin  In maturing endosome, invaginaon takes place  receptor + ligand (with ubiquin tag) enter the evaginaon area  goes to intralumenal vesicle  shuts o signaling since it’s not accessible to the cytosol  Mulvesicular body fuses with lysosomes and everything gets degraded Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 Unit 7 - Cells communicate by extracellular signaling o Signal goes to a cell  itself or far away  Signaling cells synthesize and release signals that can be small molecule, proteins, large molecules  Produce a specic response when binding to receptor molecules in the target cell - Signal transducon o Process of converng an extracellular signal into an intracellular o Can have many cellular responses Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - 6 steps in signaling o (1) synthesis of signaling molecule by signaling cell o (2) release of the signaling molecule by the signaling cell o (3) transport of the signaling molecule to the target cell o (4) detecon of signaling molecule by receptor protein o (5) a change in cell metabolism, funcon or development triggered by the receptor-signaling molecule complex o (6) removal of the signaling molecule, which terminates the cellular response - 4 types of signaling pathways o (1) autocrine  itself Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (2) paracrine  other cells o (3) endocrine  hormones  act on dierent cells via the blood o (4) contact-dependent  expand at the surface of the cell  must be neighbors  membrane to membrane contact with each other Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Extracellular signaling molecule alone is not the signal o Depends on how the target cell interprets the signaling molecule - Intracellular signaling pathways o Signaling pathways  needs proteins + second messengers o Each protein alters the conformaon/acvity of the next protein  Can also be intracellular signaling molecule  cAMP or calcium ion => second messengers o Protein conrmaon  phosphorylaon  Kinases add phosphate groups while phosphatases remove them o Receiving message to alter cell acvity Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - 4 types of molecular switches when signaling is mediated by proteins o (1) phosphorylaon or dephosphorylaon  O  kinase adds a phosphate  On  phosphatase removes a phosphate o (2) GTP Binding  G protein binds to GTP  hydrolysis occurs  GDP Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (3) assembly-disassembly of protein complexes o (4) proteolysis - Protein phosphorylaon o Changes protein behaviour in dierent ways  (1) acvate or inacvate an enzyme  (2) promote or interfere with protein-protein interacons  (3) change subcellular locaon  (4) trigger protein degradaon - GTP-binding protein is acve when bound to GTP and inacve when bound to GDP o No signaling protein  GTP-binding protein is bound to GDP o Signal  releases GDP and it binds to GTP o GTPase acvity  hydrolysis GTP to GDP  going from acve form to inacve form - Many signal-transducon cascades contain mulprotein signaling complexes o Some proteins act as a scaold  all proteins that need to be turned on are all bound to the scaold protein  Brings other signaling molecules closer for further acvaon o Receptor itself can be a scaold Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Most interacons are mediated by specic protein domains (poron of a protein that has dierent roles  funcons independently of the protein)  Examples:  SH2  binds to phosphate in a parcular sequence  SH3  binds to proline-rich sequences  PH  binds to certain phospholipids  PTB  binds to phosphorylated tyrosine residues in a parcular sequence Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Can be molecular  a protein can have mulple domains when a signal is on  each domain acts with a dierent protein o Proteolysis  intracellular poron is cleaved and acts as a signaling molecule  Switch is irreversible  once protein is cleaved it cannot be undone - Intracellular signaling pathways  cascade of events o (1) Relay the signal  spread it in the cell o (2) amplify the signal  all it takes is for one ligand to bind to one receptor  causes huge intracellular reacons o (3) distribute the signal  can have mulple dierent responses for one signal  Can have mulple pathways at one me in a cell o (4) Integrate various signals from mulple dierent pathways  using second messengers Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Extracellular signaling molecules  2 classes of receptors o 1st class (largest)  Extracellular signaling molecules are too large + too hydrophilic to cross the plasma membrane  Receptors are on the outer surface of the plasma membrane o 2nd class  Smaller hydrophobic signaling molecules where the receptors are found inside the cell - 3 classes of ligand-triggered cell-surface receptors o (1) G-protein-coupled receptors – found in virtually all cells o (2) Ion-channel-coupled receptors  neuronal signaling o (3) enzyme-coupled receptors  kinase acvity - (1) G-protein coupled receptors  2nd messenger Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (1) G-protein linked receptor binds to its ligand o (2) ligand binding  induces interacon of the receptor with inacve GTP- binding protein o (3) binding to the receptor acvates the G protein o (4) alpha subunit of the acvated G protein leaves the receptor and binds the inacve eector enzyme o (5) binding to the acvated G protein acvates the eector enzyme that generates a second messenger (usually cAMP)  When cAMP producon is smulated  Gs  When cAMP is inhibited  Gi Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Some proteins smulate the producon of diacylglycerol and inositol-1,4,5- triphophate by acvang phospholipase C  called Gq  Has Gq alpha and Gq beta subunits - Ion channel-coupled receptors o Ligand binds to the receptor (acvaon area), channel opens and ions ow in - Enzyme coupled receptor o (1) An enzyme-coupled receptor binds its extracellular ligand o (2) switches on an enzyme acvity on the opposite side of the plasma membrane  Enzyme can be a part of the cytoplasmic domain of the receptor  Enzyme associates with cytoplasmic domain of the receptor nd - 2 class of extracellular signaling molecules and receptors o Some signaling molecules  small and hydrophobic and can diuse across the membrane  Signaling receptor is found inside the cell  nucleus or cytosol o 2 types of groups of the signaling molecules menoned above  (1) Hormones  receptors = transcripon factors that regulate expression of specic genes  (2) Nitric oxide (NO)  produced by the breakdown of arginine  Short -lived  Local acng (paracrine signaling)  important in cardiovascular Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Ex 1: Corsol  produced by adrenal gland when stressed & have low blood glucose o Makes the body produce glucose and suppresses the immune system o (1) Corsol diuses across the plasma membrane and binds to its receptor protein in the cytosol o (2) binding of corsol  receptor conrmaon change  becomes the corsol- receptor complex  it is transported into the nucleus through the nuclear pore o (3) nucleus  receptor binds to specic DNA sequences and induces expression of the downstream genes - Ex 2: Thyroxine  increases metabolism and acvates protein synthesis by knocking o protein synthesis repressor o Thyroxine receptor is found in the nucleus and is bound to DNA (with or without thyroxine)  When thyroxine is not present, the receptor binds with a repressor molecule to prevent gene expression o (1) Thyroxine is produced by the thyroid gland and diuses across the plasma membrane o (2) Thyroxine gets transported into the nucleus through the nuclear pore Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (3) In the nucleus, thyroxine binds to its receptor  induces a conformaonal change where it binds with a transcripon acvator o (4) the transcripon acvator recruits RNA polymerase  gene is transcribed - Ex 3: nitric oxide (NO)  vasodilator  causes muscle cells lining the blood vessels to relax  allows for blood vessels to relax o Produced locally and goes to an epithelial cell o Allows muscle cells to chill o (1) NO acvates guanylyl cyclase which produces cGMP (second messenger) o (2) cGMP acvates a protein kinase that phosphorylates specic substrates  relaxaon of muscle cells lining the blood vessels in order for them to expand Unit 8: Part 1 - Gs-protein-coupled receptors that signal through the second messenger cAMP o (1) Ligand binds to receptor  acvates the receptor o (2) Heterotrimeric Gs protein associates with the receptor Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Gs acvated by replacing GDP with GTP o (3) alpha subunit of the Gs protein dissociates from the beta-gamma subunits  Alpha subunit associates with adenylyl cyclase o (4) adenylyl cyclase is acvated produces the second messenger cAMP with ATP o (5) turning o the signal  (a) Hydrolysis of GTP (into GDP + P) on the alpha subunit  (b) conversion of cAMP to AMP by a phosphodiesterase enzyme Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - cAMP producon o formed from ATP via a cyclizaon reacon  removes 2 phosphate groups from ATP, then joins the “free” end of the remaining phosphate group to the sugar part of the molecule o cAMP to AMP is done by phosphodiesterase enzyme  reduces one of the ester bonds Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - cAMP eects in animal cells o mediated by the acon of cAMP-dependent protein kinase (protein kinase A = PKA)  inacve form of PKA  tetramer  2 regulatory (R) and two catalyc (C) subunits  1 (R) subunit = 2 two cAMP binding sites  Therefore PKA has 4 cAMP binding sites  cAMP binds to PKA  acvates PKA  catalyc (C) subunits are released and can phosphorylate downstream target proteins  phosphorylaon done by catalyc subunits  mostly acvates downstream proteins but can somemes inacve them o ex: adrenal-mediated rise in cAMP  important with body’s response to stress (ght or heavy exercise)  all ssues need glucose and fay ssues  response of a ssue  depends on the types of receptors and signaling pathways that are acvated  adrenaline binds to adrenergic receptors  beta-adrenergic receptors  Gs acvaon  alpha-adrenergic receptors Gi acvaon - Glycogen metabolism o Glycogen  major form of storage of glucose o Glycogen to glucose synthesis  (1) glucose is coupled to UDP  glucose gets incorporated into polymer by glycogen synthesis  (2) when glucose is needed  glycogen phosphorylate (enzyme) removes sugar as glucose-1-phosphate from glycogen Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (3) glucose-1-phosphate is converted into glucose-6-phosphate gets incorporated into glycolyc pathway in muscle cells  (4) liver cells  phosphate is removed and glucose is transported into the blood  carried to other cells Muscle cell - Glycogen metabolism o (1) adrenaline binds to its receptor  receptor undergoes conrmaon change  gets acvated o (2) leads to the acvaon of the alpha-subunit (Gs alpha)  conrmaon change  acvates adenylyl cyclase  cAMP levels rise o (3) cAMP binds to PKA o (4) one of PKA’s target  glycogen synthase Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Phosphorylated form = inacve  prevents glucose producon  Can be restored to its acve form from the protein phosphatase 1 (dephosphorylates) o (5) another of PKA’s targets  phosphorylase kinase  Kinase gets phosphorylated  acvated  Reversal of acvaon is done by phosphatase 1 o (6) phosphorylase kinase phosphorylates glycogen phosphorylase  glycogen phosphorylase  iniates the removal of glucose from glycogen  reversal of glycogen phosphorylase  reversed by phosphatase 1 - PKA pathway has a fast and slow response o Fast response  signal transducon in the cytosol  Acvaon of PKA  Phosphorylaon of glycogen synthase, phosphorylase kinase and glycogen phosphorylase o Slow response  cAMP-inducible gene transcripon Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (1) catalyc subunit of PKA translocate to the nucleus  (2) phosphorylates and acvates CREB (transcripon factor)  cAMP genes have a DNA sequence upstream of the gene called CRE  (3) CREB dimerizes (doubles) and binds to CRE  (4) this binding recruits CBP and p300 (and other proteins)  modies DNA so that it becomes transcriponally acve - Turning of adrenaline/glucagon signal o (1) Anity between receptor and ligand decreases in the presence of G s alpha  limits number of Gs alpha acvated o (2) Hydrolysis of GTP on Gs alpha  increased by adenylyl cyclase o (3) phosphodiesterase converts cAMP to AMP o (4) beta-adrenergic receptor becomes a substrate for PKA and BARK (another kinase) that desensizes it - G-protein coupled receptors also signal through phospholipids o Some second messengers are derived from phosphadylinositol  (1) inositol is phosphorylated by kinases (many can do so)  cleaves at either the 4 or 5 posion of the ring  (2) phospholipase C enzyme hydrolyzes inositol ring  Releases two second messengers  DAG and IP3  (3) IP3 and DAG trigger separate downstream events  DAG  membrane bound  IP3  can diuse in the cytosol o Steps:  (1) ligand binds  acvates Gq alpha  separates from the beta/gamma subunit Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (2) Gq alpha acvates PLC-beta  releases DAG and IP3  (3) IP3 goes to calcium channels in the ER  (4) Calcium channels open  releases calcium into the cytosol  (5) Calcium binds to PKC (protein kinase C)  causes protein to relocate to plasma membrane  (6) PKC associates and is acvated by DAG  (7) PKC can phosphorylate other downstream targets - Glucose in the blood is a signal for the release of insulin from pancreac cells o (1) glucose level rises above 5mM  imported through GLUT2 transporter o (2) increase of glycolysis  produces ATP and pyruvate o (3) ATP binds to ATP-sensive-K+ channels  closes channels o (4) closing channel causes depolarizaon of the plasma membrane o (5) depolarizaon causes voltage-sensive Ca2+ channels to open o (6) increase in intracellular Ca2+ channels  triggers release of vesicles containing insulin Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Calmodulin (CaM)  binds to Ca2+ o When CaM is bound to Ca2+  binds to many kinases that have dierent responses o Acve CaM kinases can  Phosphorylate myosin light chain => converng myosin from an inacve to an acve state  Phosphorylate and acvate CREB protein  acvates CREB so that it can smulate gene expression o When CaM (bound to calcium) binds with PDE1  Increases its acvity  reduces cAMP levels in the cytosol - Regulaon of muscle contracon by Calcium and CaM o (1) A rise in intracellular calcium causes it to bind to CaM o (2) CaM-Ca2+ acvates myosin light chain kinase (MLCK) o (3) MLCK phosphorylates light chain of myosin  binds to acn to iniate muscle contracon Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Regulaon of CREB transcriponal acvity by calcium and CaM o (1) A rise in intracellular calcium causes it to bind to CaM o (2) CaM-Ca2+ acvates CaMK2 o (3) CaMK2 enters nucleus through pores and phosphorylates CREBS o (4) CREB dimerizes (doubles) and binds to CRE sequences, recruits p300 and CBP to acvate gene transcripon - Regulaon of cAMP levels by Calcium and CaM o (1) A rise in intracellular calcium causes it to bind to CaM o (2) CaM-Ca2+ acvates PDE1 (highly expressed in brain, heart and lung) o (3) PDE1 converts to cAMP to AMP  downregulates cAMP signaling pathways Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Calcium acts to inhibit glycogen synthesis and acvates its degeneraon o PKC is acvated through Gq receptor o PKC targets glycogen synthase  Phosphorylaon of glycogen synthase inhibits its acvity  inhibits glycogen synthesis o Phosphorylase kinase  4 subunits (alpha, beta, gamma, delta)  Delta subunit is CaM o Increase in intracellular Calcium  acvaon of phosphorylase kinase  acvates glycogen phosphorylase Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o With rise of calcium, it inhibits glycogen synthesis (through PKC) and acvates glycogen degradaon (through calcium) Unit 8: part 2 - Some receptors  acvated with low concentraons of ligand available o Ligands  growth factors and includes hormones o Response  takes hour since it alters gene expression  Involves phosphorylaon of a downstream target or the receptor itself Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Receptor examples  receptor tyrosine kinases (RTKs) o Tyrosine is phosphorylated  due to being involved in a signaling pathway o Responds to decrease in concentraon of growth factors (which are potent) - RTK o Has a singular membrane spanning domain o Ligand binding  induces aggregaon of receptors + autophosphorylaon (one receptor phosphorylates the other’s tyrosine) - Ras-Raf-MAP kinase pathway o Ras  small GTP binding protein  When bound to GDP  inacve  When bound to GTP  acve o GRB2  an SH2 domain-containing protein (SH2 domain  binds to phospho- tyrosine)  Mediates acon of the Sos to the acvated EGF receptor  EGF binds to EGF receptor  inhibits cascades of events  acvaon of small GTP binding protein Ras  (1) aggregaon of EGF receptors Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (2) autophosphorylaon of receptors on tyrosine  (3) GRB2-Sos complex binds to the acvated receptor through SH2  (4) Inacve Ras associates with Sos  (5) Sos acvates Ras  makes it release GDP and bind to GTP Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  (6) acvated Ras binds to Raf and the protein 14-3-3 (inhibitory protein)  allows for inhibitor to leave and for Ras to phosphorylate  (7) Ras hydrolyses  releases acvated Raf  (8) Raf acvates MEK  (9) MEK phosphorylates and acvates MAP kinases (ERK1, ERK2) o MAP kinases targets  transcripon factors  Ex: p90RSK acvated and translocate to the nucleus (with MAP kinase)  MAP kinase phosphorylates TCF and p90RSK SRF Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Ras-Raf kinase acvates early response genes (needed for the cell to enter and progress through the cell cycle) o Genes have serum-response element (SRE)  acvated by growth factors in serum o SRE binds to unphosphorylated forms of TCF and SRF  Phosphorylated  acvate transcripon o Sos  nucleode exchange factor for Ras  acvates GTPase Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Overview o Raf, MEK, ERK1/2  Most downstream  ERK1/2  MAPK  MEK phosphorylates MAPK  called MAPKK  Raf phosphorylates MAPKK  called MAPKKK - Inhibit Ras-Raf-MAP kinase o (1) GTP hydrolysis by Ras  mutaon of Ras to a form that cannot hydrolyze GTP o (2) Dephosphorylaon of MAPK  one gene whose transcripon is upregulated by ERK1 is MKP-1 (a phosphatase that acts on ERK1) Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (3) Inacvaon of receptor through:  Receptor dephosphorylaon  Endocytosis + delivery to lysosomes  Anbody inhibion  Hercepn inhibits EGF receptor - Insulin receptor pathway o Insulin receptor  heterotetramer  2 alpha and 2 beta subunits  Alpha  extracellular, form a binding pocket for insulin, covalently linked to beta subunit  beta  cytosolic, has tyrosine kinase domain o (1) binding of insulin changes conrmaon of alpha subunit conrmaon change in beta subunit o (2) brings the beta subunits closer together  acvates its tyrosine kinase acvity  autophosphorylates o (3) autophosphorylated receptor binds to insulin receptor substrates (IRS1 and IRS2)  get phosphorylated o (4) IRS1 acvates several pathways  Ras by binding to GRB2-Sos and phosphadylinositol by binding to lipid kinase PI3K o (5) PI3K phosphorylates  phosphadylinositol-4-phosphate  forms PIP2 (phosphadylinositol-3,4- biphosphate)  phosphadylinosiol-4,5-biphosphate  forms PIP3 (phosphadylinosiol-3,4,5-triphosphate)  PIP2 and PIP3 are second messengers and recruit various proteins to the membrane Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o (6) PIP3 recruits AKT and PDK1 o (7) PDK1 acvates and phosphorylates AKT  mTOR is also acvates AKT o (8) AKT dissociates from membrane  dierent pathways Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420  Protein synthesis  Glucose uptake (inducing GLUT4 transporter to the cell surface)  Glycogen synthesis - Inhibion of insulin receptor pathway o PI3K  terminated PTEN  Converts PIP3 to PIP2 - RTKs o Also acvate phospholipase C o Acvates PLC o Thus RTK can also signal through IP3  rise in calcium and acvates downstream molecules such as PKC and CaM Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Signaling through proteolysis  Notch-Delta pathway o Important for cell fate determinaon o (1) Delta = ligand while Notch = receptor  Transmembrane proteins o (2,3) when they both interreact  Notch is cleaved by:  ADAM10 (cleaves extracellularly)  Presenilin 1 (cleaves within the membrane spanning region and releases a fragment of the Notch intracellular domain) o (4) domain then translocates to the nucleus where it acvates transcripon Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 - Presenilin 1 in Alzheimer’s disease o APP neural membrane protein gets cleaved by ADAM10 and presenilin 1  yields and innocuous 26 amino acid pepde o APP neural membrane protein gets cleaved by beta-secretase and presenilin 1  yields 42 amino acid pepde  aggregates to form amyloid plaques - Convergence, divergence and cross-talk in signaling o Convergence  dierent receptors acvate similar pathways  Leads to the expression of similar genes o Dierence  A pathway that branches o from another pathway Downloaded by Elisabetta Ferro ([email protected])  lOMoARcPSD|47133420 o Cross-talk  One pathway inuences another pathway - Studying a signal pathway o (1) Protein-protein interacon  co-immunoprecipitaon, yeast 2 hybrid o (2) mutagenesis  determine key amino acids required for interacon and for signaling o (3) using bypass experiments with a mutant cell lines to determine the order of the pathway Downloaded by Elisabetta Ferro ([email protected])