Class Notes (838,933)
Canada (511,158)
ANAT 212 (5)
Lecture

ANAT 212 - Teacher 1.docx

51 Pages
158 Views
Unlock Document

Department
Anatomy & Cell Biology
Course
ANAT 212
Professor
Thomas Duchaine
Semester
Winter

Description
 Cytosol is packed with proteins and macromolecules  -Proteins are made as linear polypeptide but must fold into 3D conformations in order to be functional o Folding makes the protein more physically stable and functional  peptide bonds in the back bone become uncharged when the amino acids are linked into polypeptides o Charge and hydrophobicity of a polypeptide is determined by the side chains o Both 1) side chains and 2)backbone can form non-covalent contacts with other amino acids  peptide bond is planar and cannot rotate o You can only rotate around the central carbon(Ca) o Polypeptide backbone has limited freedom of rotation o Some rotation angles between amino acids (residues) in a polypeptide are preferred Non-Covalent Bondsinteraction between residues stabilize the folding! – hydrophobic interactions (exclusion of water) – hydrogen bonds – van der Waals interactions (transient dipoles between all atoms) – ionic bonds[+ve charged group interacting with –ve) hydrophobic interactions very many, strong hydrogen bonds many, strong Van der Waals interactions many, weak ionic bonds few, strong disulfide bonds few, very strong  Folding is driven mostly by hydrophobic interactions! o The other non-covalent interactons contribute to the stability o Native State is the most stable conformation o Proteins with similar sequences usually have similar native states, and may have similar functions  Secretory proteins often have covalent disulfide bonds between cysteine side chains o Only in places wit oxidizing environment: 1)extracellular proteins(protein that are to be secreted outside the cell) 2)secretory organelles  Cytosolic proteins do not hav disulfide bondstheyre in reducing environment: cytosol, nucleus, mitochondria  Hydrophobic interactions(in the core)  ionic bonds(on the surface) Loopsnot regular secondary structuresflexible Secondary StructuresAlpha helices & Beta sheets stabilized by H-bonds Tertiary Structuresvariety of secondary elements  stabilized by Hydrophobic interactions! Quaternaryoligomer: many subunits(more than dimer,trimer,tetramer,5-mer,6-mer…) -In tertiary structure the residues in the primary sequence that make contact are far apart from each other -A domain is an independently folded unit within a protein -protein functional surfaces can interact with ligandsHighly specific protein recognizes fewer molecules -Allostery: conformational changes can change binding surface -Modular domains are domains that are found in many different proteins, thus its function can be found in different proteins -Many modular domains form reversible, non-covalent contacts with specific features on other proteins, lipids, carbohydrate, and allow regulation of function -Similarity (homology) indicates evolutionary conservation -Divergent sequences have no similarity and different structures, but may have related functions[structure and function is not always conserved together] -Folding is thermodynamically favoured (negative DG free energy) -Folding can be spontaneous, even though its helped by mechs(chaperones) -Native structure is determined by the primary sequence -Native state is more packed so will take u less volumes compared to an unfolded protein Unfolded (denatured) domains are very flexible and have NO secondary or tertiary structure - As it folds it Increases in secondary structures and gets to the native state -Molten Globule are intermediates that are close to native -Molten globule has little 1)secondary structure elements and are 2)still flexible compared to native state… -Native State structure is stabilized by hydrophobic interactions(Tertiary structure) -Some domain require a ligand in order to function!!(another protein subunit or cofactors) -Some domain do not always however still function to bind ligand reversibly -Native state can be in equilibrium with near-intermediates hydrophobic residues are scattered throughout the primary sequence… allowing them long distance interaction where these hydrophobic residues come together -Risk of Aggregation: where more than one unfolded proteins are present, and come together forming hydrophobic interactions, that eventually cannot be separated, and cause biological problems Incompletely folded proteins: 1)present right after synthesis 2)when the protein’s ligand is not available due to problem 3)a folded protein that faces stress stress(heat) Folding “Quality Control” -during folding process, the intermediate can be off-pathway, -Quality control mechs correct misfoldings 1)defect is corrected through molecular Chaperones 2)Proteosome degrades defected protein 1)Chaperones Chaperones help folding & prevent aggregation, without being part of the final native state of the substrate -Folding intermediates have hydrophobic patches that are exposed… this is what the chaperones recognize -Heat Shock Proteins = HSP (HSP70… weighs 70kDa) -Chaperones are also essential under non-stress conditions -Some substrates are folded by a specific chaperone or combination of chaperones -Some substrates are folded without the help of a chaperone ATP-independent chaperones prevent aggregation since they cover the exposed hydrophobic patches, AND they can catalyze some folding steps but not as efficiently as an ATP-dependent chaperone • both ATP-dep & ATP-indep can cooperate to work Families of Chaperones {Coevolution of chaperones with substrates depending on organism} Families have similar mechanism & structure 1)HSP70  (found in Bacteria, {Human Cytosol, ER, Mitochondria} 2)HSP60(Chaperonin) (found in Bacteria, Archaea, {Cytosol, Mitochondria} 3)HSP90  (found in Bacteria, {Human Cytosol, ER, Mitochondria} • Rotation around backbone is slowed by large side chains, • Slower rotation causes slower folding • Due to extra covalent bond, Prolines rotate around backbone the slowest thus folds slower • Protein with Tryptophan is slowly folded as well due to large side chain • Glycine is quickly folded [due to no side chainonly an ‘H’] Peptidyl-prolyl isomerases (PPIases) ATP-independent chaperone** recognizes and increases proline rotation and can speed up folding 1)HSP70 Family[monomers?homodimers?] • Work as Monomers: single polypeptide with no quaternary structure • Has Two Domains i)Peptide binding domain ii) ATPase domain two-state mechanism is highly conserved between organelles and organisms ATP-bound  there is no substrate binding [both domains tighten] ADP-bound  tightly binds substrate Substrate binding domain recognizes short(7AA) polypeptide domains that are hydrophobic and have an extended unpacked conformation - HSP70 being a monomer that recognizes short sequences, i) there can be more than one Hsp70 binding sites in one polypeptide, AND ii) many diff. polypeptides can be bound by HSP70 since the recognized sequence is short and possibly common DNA-J Co-chaperone [HSP40] HomodimersAlso recgnizes short hydrophobic sequences like HSP70 i) J-domains are modular domains, can be found in other proteins, but always function to bind to HSP70 and activate hydrolysis of ATP -Jdomains never bind the substrate, -however, DNAJ’s have another separate domain that is a ii) substrate-binding domain which acts as an ATP-independent chaperone -Also has iii)Dimerization domain that joins another HSP40’s Dimerizing domain to form homodimers DNA-J proteins can be classified: Type 1 DNA-J : Have conserved Substrate binding domains Type 2 DNA-J : Have divergent substrate binding domains Type 3 DNA-J : NO SUBSTRATE BINDING DOMAIN ONLY Type 1 and some Type 2 act in folding HSP70 NEF Co-chaperones [Nucleotide Exchange Factors] -Opens ATPase domain to promote release of ADP to replace it with ATP -causes release of substrate from HSP70 HSP70 Cycle -HSP40 binds a substrate, -HSP40 interacts with HSP70 (to stimulate hydrolysis of HSP70’s ATP) -HSP70 takes over and grabs substrate as ATP is hydrolyzed [HSP40 transfers substrate onto HSP70] -HSP40 leaves -NEF comes and replaces ADP, -HSP70 opens up and releases substrate as an unfolded protein that will be folded to (i)its next intermediate form through another HSP70 or (ii) to its native state through the next chaperone/chaperonin -HSP70 is available to repeat process DNA-J Co-Chaperone is ALWAYS required for HSP70 [Transfer of substrate from HSP40 to HSP70 is a critical step] NEF importance varies…not always required 1)HSP60 Family {CHAPERONINS} HSP60 have multiple subunits (UNLIKE HSP70 that are monomers) HSP60 family all have double ring structure (e.g. E.coli GroEL) Sometimes Chaperonins involve a co-chaperone(e.g. E.coli GroES cap) GroES sits on top of GroEL enclosing substrate within cavity GroEL: made of 2 rings, EACH ring with an i)ATPase domain and an ii)Apical domain Each GroEL ring’s ATPase domain is facing eachother and are at the interface of the two rings and the Apical domains facing away from eachother -????”?Movement of the Apical domain is controlled by nucleotide in the same ring(top) and opposite ring (bottom)???????? -- GroEL Apical Domain Slide -- GroEL Cycle Slide -- Human Hsc70 acts at early intermediate stages of a folding protein, when hydrophobic patches are exposed… Human chaperonin TriC acts after Hsc70 at late folding stages close to native state TriC is specific for certain substrate protein -Both Hsc70 and TriC make no direct contact, Hsc70 just does its job and lets go of the folding protein, which is then grabbed by the TriC which helps it fold further Chaperonins are large, but only hold substrate of certain size… if TriC is 700kDa it Can’t hold 700kDa substrate 3)HSP90 Family P23 is co-chaperone ONLY human cytosol version of the HSP90 HAS a Co-chaperone Human ER, mitochondria, and bacteria cytoplasm DOES NOT have a Co-chaperone UNLIKE HSP70 where they require the Co-chaperone in order to work… here HSP90 doesn’t need the co- chaperones however, the co-chaperone increases its efficiency the ‘no direct contact’ of the Hsc70 vs TriC contrasts the HSP90 family HSP90 chaperones are homodimers (the 2 subunits are joined at C-terminal) ATP controls opening and closing of dimer SINCE IT IS THE ATPase DOMAIN THAT OPENS AND CLOSES and TRAPS THE SUBSTRATE IN Has i)ATPase domain and ii)dimerization domain Binds near-native state(late stages of folding) molten globule intermediates HSP90 cycle -When there is NO nucleotide(nucleotide-free state) HSP90 is open -When ATP is bound, HSP90 dimer closes up  Substrate can be bound at BOTH i)nucleotide-free state and ii)ATP-bound state -when ATP hydrolyzes, HSP90 compacts the dimer even more, this releases substrate -Then when ADP is released, HSP90 opens up for another substrate to come in during its nucleotide-free state Multichaperone System HSP70 & HSP90 can co-operate  different from HSP70 & HSP60 cooperation Co-chaperones make direct contact to these chaperones and Many Co-chaperone exist in regulating HSP70&HSP90… therefore these co-chaperones have modular domains, so some proteins can have these domains(thus folding function) while also have non-folding function! HOP Co-chaperone binds Hsc70 and HSP90 at the same time! Even though Hsc70 and HSP90 are different families with unrelated structure, they both have similar C- terminals sequence motifs EEVD HOP has TPR domains that are the regions that recognize the EEVD sequence motif Note: E and D have acidic side chains that are charged &&& V is hydrophobic Pattern of charges with the Hydrophobic Valine in between is what is recognizes by the HOP co-chaperone Note: the bonds with HOP are ionic(due to the charges), hydrophobic, and hydrogen bonds HOP: has 2 TPR domain(one to bind HSP90 and one for hsc70) CHIP: has 1 TPR domain(binds EITHER hsc70 or hsp90) and has 1 U-box domain(for ubiquitin ligase) FKBP52: has 2 PPI domains & 1 TPR domain(binds ONLY Hsp90) Note: TPR domains are not dependant on nucleotide state of Hsc70 or Hsp90!!! Human Hsp90 ATPase cycle(ATP cycle opposite of HSP70, where ATP-bound state for Hsp70, NO substrate!) -start with hsp90 in nucleotide free state and binds to Hop -Hsc70 binds substrate and gets binded to Hop and transfers the substrate onto Hsp90 that's also on Hop -When hsp90 binds an ATP, it closes onto the substrate[while p23 helps hsp90 stay closed] -Hop and Hsp70 disassociate -When Hsp90 hydrolyzes ATP to ADP, it compacts even further, -The substrate and p23 eventually disassociate -These are same are common ATP cycle steps except Hsp70 and Hop bring the substrate to Hsp90 Glucocorticoid Receptor GR is a hormone receptor When bound to cortisol, it responds by activating/repressing Genes i) Ligand-binding domain binds Cortisol hormone in the interior hydrophobic area, this hormone is a ligand that is necessary to make the LB-domain fold stable for GR’s total structure to be stable for function… The rest of the domains are already fully folded and are already stable! ii) DNA-binding domain binds GR promoter elements(GRE) iii) N-terminal activation domain regulates transcription LBD is folded by the chaperones: 1)hsp40, brings LBD that is on the GR, to hsc70 2)hsc70 takes LBD and gets bound to hop’s TPR domain 3)together hop also binds to hsp90 with the other TPR domain 4)hop and Hsc70 hand over LBD 5) hop and hsc70 leave 4)p23 cochaperone stabilizes and helps hsp90 stay closed 5)FKBP52(steroid receptor chaperone) helps hsp90 fold the GR(steroid receptor) by inserting a hormone ligand(cortisol) into the hydrophobic interior that is exposed.. if there is no presence of hormone then the GR goes back to step 1 and continues cycle…WITH HORMONE,the GR becomes a Dimer and is Active Heat Shock Response After heatshock there is a recovery period(1h to 24h) -immediately after heatschok Translation is inhibited, and it slowly recoveres Increased chaperone expression & increased degradation of unfolded shit Increased Quality control factors -Transcription of HSP’s are up-regulated after couple of hours, while transcription of other proteins are down-regulated These Heatshock response is regulated by HSF1 Transcription factor HSF1 has i) DNA-binding domain ii) Trimerization domain iii) Transcription activation domain HSF1 is inactive in its monomeric form Active in Trimer form! ONLY when active, HSF1’s DNA-binding domain recognizes HSE promoters (heat schock element) And HSF1’s Transcription activation domain regulates the transcriptions that occurs for Heatshock response Chaperone mechanism is essential to the regulation of HSF1 and shit -HSF1 in its monomeric form is in native form, but MIMICS unfolded proteins and is bound by HSP90 chaperone in normal daily circumstances -however, after heatshock, the HSP90s tend to let go HSF1 and go fix the actually unfolded proteins instead -by leaving the HSF1’s it allows HSF1’s to Trimerize and activate heat shock response transcriptions! -However, Negative feedback occurs, since due to Heat shock response, chaperone expression is increased, this includes an increase in HSP90, so after HSP90 does its shit and all the denatured proteins are folded up, -this leads to the end o the recovery period, where there is an excess of HSP90’s -This excess causes them to bind monomeric HSF1,(HSF1 trimerized form is equilibria to its monomeric form) -Binding to monomeric HSF1 causes them to be kept away from being able to trimerize! Thus downregulation! Protein Modification After translation, proteins are i)cleaved by peptidases ii)**COVALENT** modifications of N-terminus modifications can change conformation of protein, or create/block binding sites , and can be used as switches to turning on and off certain functions Serine(S), Threonine(T), Tyrosine(Y) Phosphorylation KINASE takes Phosphate GROUP(PO ) &3replaces “H” from Hydroxyl group(OH) of each {S,T,Y} Serine + Phosphorylation of OH = Phosphoserine Same for Threonine… Kinase puts phosphate group on the Serine or Threonine or both Since Tyrosine is much more different in structure compared to {S,T} so they’re modified by a different Kinase! Phosphorylation increases Size and Charge! Lysine(K) , Arginine(R) Acetylation & Methylation Acetylation changes polarity(charge) and Size Methylation doesn't affect charge as much, but increases hydrophobicity(adds more Methyl), and Size So they can be used to activate diff. signals and binding interactions • Transferases that put on the Acetyl- and methyl- are sequence specific [there are also pressne of deacetylases and demethylases that remove the modification] Lysine (K) Ubiquitination Ubiquitin’s C-terminus is Covalently linked to Lysine’s N-terminus, forming a covalent ‘peptide bond’ Ubiquitination ONLY on Lysine(NH3+)Not Arginine(NH2+), Not Histidine(NH+) -many Lysines can be ubiquitinated, but not all… depends on accessibility and E3 Ub’s C-terminus can be attached to another Ub and form Polyubiquitin chains -Poly Ubiquitin on Lys48 Degradation via proteasome -Poly Ubiquitin on Lys63 DNA repair & Endocytosis Asparagine (N) Glycosylation -N-linked glycosylation only on Asn side chain’s amide N-link only occurs on Asn-X-Ser/Thr motif [Check N-linked Glycosylation paragraph for details] -ONLY ONLY ONLY Asparagine… Glutamine’s(Gln) amide is not even recognized -Glycans that are added through glycosylation can be modified after addition, but are never removed unless protein is degraded Protein degradation Quality controlChaperones & degradation PREVENTS AGGREGATION -Part of Quality control Ubiquitin mediated degradation by Proteosomes in cytosol Ubiquitin Mono-ubiquitination is a tag for Localization of proteins within the cell Polyubiquitin tag for degradation by proteasome Ubiquitin is not degraded itself but is recycled Process starts with E1 ubiquitin activating enzyme that binds itself to Ub and transfer it to E2 ubiquitin conjugating enzyme in a chemically reactive state E2 will do the attachment of Ub onto the substrate polypeptide E3 Ligase enzyme provides specificity and brings the specific substrate that will be modified by Ub **There are NOT much E1& E2 enzymes, but MANY different E3 ligasewide range of substrate specificities Degradation is controlled by E3, not the proteosome!! proteosome just destroys what is tagged …E3 is what tells the E1&E2 what substrate to ubiquitinate. The Proteosome Protein degradation in i) Cytosol and ii) Nucleus & unfolded shit in the iii) Endoplasmic reticulum Core+Cap =(26S) proteosome Has Central Core (20S) and TWO identical Caps (19S) at each end Core (20S)= 4 rings 2 outer rings Alpha subunitsProvide binding site for 19s cap 2 inner rings Beta subunitsProtease activity from inside! Cap (19S)= Base + Lid Lid: Non-ATPase Non-ATPase i) Poly-Ub receptors to recognize Poly-Ub signals on proteins ii) Ub Hydrolases that removes Ub from polypeptide Base: 6 subunits  ATPase breaks ATP in order for unfoldaseunfolds proteins and feeds em inside core Now the proteins that enter will face the 2 Beta subunits of the inner ring(protease activity) and will be cleaved at 3 different sites i) Basic sites, ii) Acidic sites, and iii) Hydrophobic sites Degradation Situations Each controlled by diff. E3 Ligase enzyme recognition mechs 1) Quality control 2) Constitutive(normal) degradation of protein that is in NATIVE state.. in order to control its level/population 3)Degradation in response to Signal 1) Quality control: Misfolded protein degradation  CHIP CHIP co-chaperone: i) TPR domain(binds either Hsc70 & Hsp90 in human cytosol) ii) U-box(binding site for E2 conjugating Enzyme)- Ub will be bound to the E2… E2 binds the U-box The substrate that is bound to the chaperone(Hsc70 or Hsp90) coming into the TPR domain will be selectively ubiquitinated!!! Chaperone[Hsc70 or Hsp90] and CHIP with E2 form a complete complex that ALSO has E3 ligase activity, therefore can ALSO select substrate to be Ubiquitinated JUST LIKE E3 ligase enzyme could. [it says Chaperone-bound substrate is selectively ubiquitinated..?? BUT where does E3’s selectivity come into play????] -E3 ligase enzymes can bind misfolded substrates directly, without Chaperones(endoplasmic reticulum) unliked CHIP complex….so does that mean that proteins can be sent to the Proteosome via E3 and CHIP complex Balance of chaperone-mediated folding and degradation if substrate is bound by Hsc70 or Hsp90 for too long, it will be taken up by CHIP and be Ubiquitinated -CHIP also competes with other Co-chaperones(Hop) that also have TPR domain that binds chaperones 2) Constitutive degradation: N-End RULE All proteins have Met in N-terminal, but they can be processed and get different residue as N-terminal -Certain N-terminal residues are considered Degrons, Degrons are recognized by E3 Ligases(UBR/recognins) with degron-binding domainsDegrons are Ubiquitinated for degradation -Recognins don't care whether the substrate is folded nicely in native state or not [disregards Quality control] Two groups of Degrons • Basic residues: Arg, Lys, His • Large hydrophobic residues: Phe, Trp, Tyr, Leu, Ile[note: Valine only has 1 less Methyl group than Leu & Ile, HOWEVER Valine is still not hydrophobic enough to be a degron!!] Some enzymes recognize N-terminal residues, and modify them into degrons Asp & Glu (acidic) are recognized and get an Arg attached onto it[as the new N-terminal residue] and becomes Degron Asn, Gln (amides) converted to Asp, Glu  Then Arg is added to them becomes degron 3) Regulated Degradation: SCF E3 Ligases(a complex) E3 ubiquitin ligase complex (Skp1/Cullin/F-box) Large complex with different parts One end grabs E2E2 brings Ub Other end grabs F-Box protein F-box protein selects and grabs substrate (diff. F-boxes can replace eachother on that end of the Scaffold, therefore replacing specificities) F-box recognizes and binds Phosphorylated peptide sequences that are on the substrate(if the substrate is not phosphorylated then it is NOT recognized) Protein Kinases can trigger ubiquitination! Phosphatases can prevent degradation through de-phosphorylation Since E2 on one end has Ub, and F-box has phosphorylated substrate on other end… This complex allows the Ub to ubiquitinate the substrate easily Theses SCF ligases degrade native proteins in order to regulate and stop their function Membranes -Some organelles and the Cell surface(plasma membrane) are connected to the secretory pathway(ER) (transport system) -Mitochondria organelle is NOT connected to secretory pathway(therefore NOT ALL organelles are connected to secretory pathway) Secretory proteins are made at the ER, and transport to Golgi, Plasma membrane -can be brought from PM to endosome or to lysosome to be degraded -(INTERIOR) Lumen of secretory pathway, vesicles and organelles are similar and equivalent to EXTRACELLULAR space  whatever aqueous environment is in organelles and secretory pathway does not mix with the cytosolthese intracellular environment provide sites within cells for biochemical reactions(i.e. photosynthesis, oxidative phosphorylation) cytosol lumenal / extracellular + + high K high Na 2+ 2+ Mg in complexes free Mg 2+ 2+ almost no free Ca Yes free Ca organic anions(organic acid) high Cl- (phosphates, carboxyls) reducing ER: Oxidizing (helps form disulfides) pH 7.2 extracellular pH 7.4 (mitochondrial matrix endosomes pH 6.0 pH 7.5, proton gradient) lysosomes pH 5.0 Fluid mosaic Model • Hydrophobicity acts as a barrier to water-soluble molecules • Membrane proteins can rotate and diffuse(move) laterally in the fluid bilayer but only in one axis, no 3D rotation • The major lipids in membranes are: – phospholipids – in all membranes – glycolipids – only at plasma membrane – Cholesterol(different form phospholipids/glycolipids) – Other types in specific organelles with special functions -Type of lipid determines mobility (diffusion, rotation) speed, and physical properties(thickness) 1) Phospholipids Polar head group: A charged group is attached to phosphate group! (e.g.Choline or other charged groups) Phosphate group is attached to Glycerol Glycerol ends the polar head group by binding to three fatty acids that form the non-polar side Polar head group is what classifies phospholipids charged groups that bind the end of the head group  i)phosphatidyl-choline[PC], ii) – phosphatidyl-ethanolamine(PE), iii) phosphatidyl-serine[PS] -Phosphatidyl-inositol (PI) is not abundant but can be phosphorylated and act as a signaling molecule -Sphingomyelin (SM) is not a phospholipid due to varied version glycerol.. but is similar… has Choline side group, just like phosphatidyl-choline -Head group size and charge affect lipid mobility Fatty acids tails -Unsaturated ones have double bond thus are going to have fixed bends thus are not flexible -Unsaturated ones Also decreases mobility(rotation and shit) due to movement-restricting double bonds 2) Glycolipids Found ONLY on outside surface of plasma membrane(PM) Head groups contain different Sugar groups NO phosphates, -Glycolipids do not have glycerol, but instead have the same varied version of glycerol as Sphingomyelin (SM) 3) Cholesterol Structurally unique, where it has four ring steroid structure that makes it hydrophobic and rigid because its structure is rigid flat and not flexible which makes mobility and rotation very slow, -Because of this, Cholesterol affects adjacent phospholipids by being in between and preventing mobility and thus reduces fluidity The polar group of cholesterol is a SINGLE Hydroxyl (-OH) group Membrane Lipid Asymmetry each sides of the membrane, exterior vs. interior are different in composition -INTERIOR has much stronger negative charges aligning the membrane due to phosphatidyl-serine! -EXTERIOR has Glycolipids Exterior: i) phosphatidyl-choline[PC], ii) Sphingomyelin (SM) iii)Glycolipids Interior: i) phosphatidyl-choline[PC], ii) phosphatidyl-ethanolamine(PE), iii) phosphatidyl-serine[PS] Interior: Low amount of Phosphatidyl-inositol (PI) for signaling purposes Organelle Lipids -Lipid composition varies between organelles -Plasma membrane has highest level of cholesterol, -Plasma membrane has highest Sphingomyelin (SM) compared to the ER or the mitochondria -Endoplasmic reticulum (ER) and mitochondria have higher levels of phospholipidsi) phosphatidyl- choline[PC] && ii) phosphatidyl-ethanolamine(PE), Lateral organization of lipids(based on hydrophobicity&Length) Mmbranes are not only organized depending on the compartment, but also organized laterally into regions(patches/microdomains) Lipids with longer tails, tend to cluster with lipids with longer tailsmaking up thicker membrane & lipids with shorter tails cluster with other short tailed lipids Cholesterol tends to cluster with the thicker membrane/longer tails,Cholesterol straightens it up Lipid raftsarre microdomains found in PM and Golgi enriched in cholesterol and thick rigid membrane Lipid rafts found on the membrane surfaces also contains diff. proteins with functions such as signaling and interaction with extracellular space, and in terms of transport into the interior lipids are not just released into the cytosol.. there are means of transport to diffuse the shit though the cytosol cus the hydrophobic lipids are not just gonna be left exposed There are also contact sites between organelles, (e.g. when mitochondria touches the ER at contact sites, lipids can be exchanged from one to the other) Phospholipids are synthesized at the ER on the cytosolic side of the ER membrane Then move through the secretory pathway to the other organelles 1) Fatty acids have a Hydroxyl group that reacts with CoA transferase which takes Coenzyme A and replaces the hydroxyl groups of the Fatty acid to make the fatty acid reactive, 2)Acyl transferase will take Two fatty acids that have been made reactive through CoA, will reactive with the two hydroxyl groups of a Glycerol-3-phosphate and make Phosphatidic acid(PA) (note: glycerol has its third hydroxyl bound to phosphate group) 3)Phosphatase will remove the phosphate group of the PA makes diacylglycerol(DAG) 4)This DAG will get its appropriate side chain through corresponding enzyme (e.g. Choline phosphotransferase which takes a CDP-choline [which is choline&phosphate together] and put it on the spot that the phosphate group used to be on, which gives off Phosphatidylcholine[PC] There are parallel similar pathways to make the other charged head groups[e.g. PE, PS] Coenzyme A -It has parts which are: ADP, with some kind of a linker on it[either Acetyl groups or Fatty acids as shown in phospholipid synthesis] that is covalently linked with the reactive Sulfhydryl group Since the sulfhydryl group can link to a variety of things, it widens the amount of function possible -The Sulfhydryl group can be linked to another fatty acyl or Acetyl • Fatty acyl CoAoxidative break down of fatty acid to acetyls in mitochondria • Acetyl CoAcitric acid cycle oxidation in mitochondria Lipid Synthesis -Phospholipids and cholesterol are synthesized on the cytosolic side of the ER membrane -Since the phospholipid will accumulate much more on the cytosolic side, an Enzyme called: Scramblase that is found in the ER will flip the lipids of the bilayer membrane RANDOMLY, leading to symmetrical growth on both halves of the bilayer Scramblase is ATP-independant -Flippase enzymes is ATP-dependant, it uses ATP to SPECIFICALLY target the flipped lipids and flip them accordingly to maintain membrane asymmetry[each sides of the membrane, exterior vs. interior are different in composition] -Non-vesicular lipid transportPhospholipid Transfer Proteins[AKA Exchange proteins] will recognize PC and the fatty acid tails of lipids synthesized in the ER and transfer them WHILE COVERING the hydrophobic fatty acid tails through the cytosol and drop them into the Mitochondria membrane mitochondria are not connected to secretory pathway by vesicle transport Membrane proteins -The sequence of a protein determines its function and localization[ALSO FOR MEMBRANE PROTEINS] -Membrane protein make contact with lipids in membraneHydrophobic contacts -membrane proteins that reside in each organelle will vary!(membrane protein of ER is not same as one in PM) -The cell has mechanisms that sort out these proteins -Integral membrane are anchored, and interact with interior of lipid bilayer -you can have transmembrane alpha helices, or beta sheets(Transmembrane Beta-barrel) spanning through the membrane -you can also have Amphipathic alpha-helix in one of the sides of the bilayer membranehydrophobic on one side and polar on the other Lipid Anchored: Proteins are covalently linked to lipid or fatty acid groups[strength depends on how many lipid are attached and the length and type of the lipid] Peripheral Membrane Protein(not as strong as Lipid anchored]: can attach through non-covalent interactions to protein&lipid i) integral membrane protein[Strong] &&& ii) lipid head groups[weak] Transmembrane Alpha-Helix (TM helices) -Also have hydrogen bonds btween the coil, side chain point outward therefore ARE hydrophobic to interact with hydrophobic interior -In the Primary sequences of TM helix proteins, there are hydrophobic residues more clustered in certain areas rather than the hydrophobic residues being scattered around… • TM alpha helices can often be predicted from hydrophobicity of primary sequence in Soluble protein, the scattered hydrophobic residues come together in Tertiary structure to become Native -Single TM helices(Single-pass) can have more than 1 helices around(Multi-pass) TM helicase are usually 18-24 amino acids longthis amount gives typical thickness of biological membrane what does he mean??? -Length of TM helices matches thickness of the membrane • Longer TM helices sit in thicker microdomains, or insert at angle in thinner membranes • Proteins with single TM helix can rotate easily! NOT FLIP Multiple TM helices can form a transporter protein(e.g. Channel proteins), leaving a space in between them forming a water-filled interior region  having one side hydrophobic and the other side polar the interior will be polarallowing charged particles across the membrane -opening is controlled by cytosolic domains or subunits – allostery Many transporter proteins have multiple TM helices, BUT the helices are NOT related (Not homologous), and just having common transmembrane features Transmembrane Beta-Barrel -TM protein formed by Beta-sheets wrapped into a cylinder -with hydrogen bonds between each strandsholding each strand together -Number of TM strands can vary and make the opening larger! -Side chains point out into lipid bilayer, AND INSIDE Membrane Protein Asymmetry -Whatever is on the cytosol side stays there and the shit sticking in lumen side stays there -The orientation of the TM protein is chosen at the time of insertion into membrane -Secretory pathway: ER have luminal/extracellular domains modified differently from cytosolic domains -disulfide cysteines only found in extracellular sideNever on cytosol side of membrane -Oligosaccharides(includes all proteins that went through Glycosylation!!!) only found in extracellular sidenever on the interior other protein modifications that are only found on cytosolic domainsi) phosphorylation, ii) ubiquitination, iii) acetylation • **Membrane proteins never flip, unlike lipids**[BECAUSE of its size, since folded protein is much larger than a single phospholipid… and also because of properties that can only be found on one of the sides, i.e. Oligosaccharides are never on the interior layer] Lipid-Anchored Proteins Cytosolic proteins that are covalently linked to lipid or fatty acid chains or Prenyl chains that are part of the Lipid bilayer membrane -Single lipid chain DOES NOT provide long lasting anchoring for the protein -Two or more lipid chains are needed for strong membrane anchoring Like phosphorylation and Ubiquitination, the attachment of these proteins are mediated by specific enzymes they attach Lipid to N-terminus or MORE OFTEN attach the Cysteine side chains of the protein for anchoring Lipid modifications (like phosphorylation but with a lipid) S-palmitoylation: i) take S-palmitate(fatty acid with Sulfide) and through a thioester bond it links it to a Cysteine side chain Reversible so by adding or removing Palmitic acid the cell can control whether the protein sticks to the membrane or notlike phosphorylation which controls protein-protein interaction Rest of the modifications are permanent, Non-reversible:  All on Cysteine side chains!! ii) N-palmitate(which is fatty acid but with Nitride instead) which forms amide bond which is much harder to break than a thioester bond iii) Myristate Also forms amide bond Prenylation iv) Farnesyl && v)  Geranylgeranyl Both are considered Prenyl lipid contain branched double bonded units throughout the fatty acid chain even though they form Thioester bond, they are irreversible Cysteine (C) are important linkages in the following schemes: • Disulfide bond formation • Lipid modification[mentioned above reversible or permanent] • Used by E1 activating and E2 conjugating enzymes to activate Ubiquitin before transfer Attachment of lipid depends on sequence of the protein around the attachment site 1) S-palmitoylation recognizes various motifs that also includes Cys in it! 2) Prenylation takes place at this motif: Cys-aax-carboxyl of polypeptide (a=aliphatic side chain, x=any side chain) when enzyme recognizes this motif, it will take Prenyl lipid and modify the ‘Cys’ side chain shown on the motif above^ Note that these motifs are not exact sequences but just Patterns Another Lipid Anchor: GPI-Anchored Proteins -there are Transmembrane proteins that are signals -these TM signal have proteins attached to them only on the Lumen/extracellular side the signal tells the proteins in the lumen surface to be cleaved and covalently link to Glycosyl-phosphatidyl-inositol(GPI)Strong attachment onto membrane this linkage allows rotational freedom since the attached proteins is on the lumen side floating around Lipid Rafts Lipid raftsarre microdomains found in PM and Golgi enriched in cholesterol and thick rigid membrane shorter membranes are excluded Contains many GPI anchored Contains Acylated proteins Excludes Prenylated protaqeins due to short length Microdomains contain proteins with similar function e.g. at the PMLipid rafts contain cluster of receptor signaling proteins e.g. at organelles sites for vesicle transport like on the endosome • Microdomains are held together by non-covalent interactions • Thus microdomains are NOT permanent, can fuse and break apart Cortical Cytoskeleton Cortical cytoskeleton is a physical structure that connects membrane to cytoskeleton of the cell  supports the PM on the CYTOSOLIC SIDE controls shape of the cells Transmembrane proteins called Glycophorin will anchor into points on the membrane Glycophorins interact with long fibres called Spectrin which are on the cytosolic side of the PM -Glycophorins and Spectrin connect to Actin fibres in the cytosol in order to connect the membrane to cytoskeleton of the cell Lateral motion  the fluid mosaic model is not exactly what occurs in realitybecause lateral movements of TM proteins is partially restricted within the membrane –i) through interaction with Actin cytoskeleton - ii) due to microdomains which only diffuse(move) around its own region(e.g. Lipid rafts only move withi Membrane Biogenesis -Cellular membrane’s can only be made by expanding on already present membranes, you cannot create it from scratch, also if a mitochondria is missing, the cell cannot make back the mitochondria from scratch -Proteins are transcribed in nucleus and translated in cytoplasm but must be sorted out to their correct compartment/membrane, and this sorting is determined by the proteins sequence and structure -there are transport mechanisms that read this information and convert it to a transport process to bring the protein to where it should work -proteins are made and go through the ER to be secreted through vesicles to other organelles Target Signals Sequence of protein can encode a signal or several signals to specify its organelle localization -often the only thing a signal does is to specify its location and not encode for the proteins function itself… this targeting signal is normally removed by proteolysis after it reaches its location. -often the signal is not an exact sequence motif but instead just a pattern which are recognized by the transport machinery -Different organelles have different mechanisms but have common features: 1)signal in protein is present and is recognized and distinguished from other sorting signals 2)signal recognition connects the protein to wherever its supposed to go 3) a mechanism is present to move the protein across to wherever its supposed to go Secretory Proteins and ER -All secretory protein go through the ER -Rough ER: Ribosomes are attached(this is why its rough), and is the site of membrane protein synthesis -Smooth ER: NO RIBOSOMES, and is the site of Lipid(phospholipid) synthesis Proteins and lipids are s ynthesized in different areas of the ER Signal Hypothesis BLABLABLA -Secretory were already known to enter ER during translation through the attached ribosomes -Observed: that newly translated secretory proteins were larger than its final form -Hypothesis: extra sequence ws a targeting signal protein that functions to direct insertion into ER membrane to do this you must have – a mechanism to read out this signal, and connect the signal to the membrane, and a mechanism that keeps the ribosome attached to the membrane – a mech to remove signal peptide Secretory Signal Sequences -These Signal sequences have a typical pattern of amino acids(not an exact amino acid sequence) -Pattern in a hydrophobic region in the linear protein sequence, usually 8 or more residues long and has short polar regions on each side of the protein sequence that makes up the pattern altogether 1)Signal sequences are at the N-terminus and are cleaved off after translocation 2) in some cases like for TM helices, the signal sequences can be in different places in the protein and become part of the TM helices so they are not cleaved off 1)-some signal regions hve shorter patterns of hydrophobic regions(8-16 residues) 2)& some lik TM helices have longer patterns of hydrophobic regions18-24 residues which is nearly the length of a TM helix, thus the signal serves as the helix itself and is thus termed: Signal anchors OVERVIEW 1) Through Peptidyl-transferase reactions, ribosomes translate the polypeptide with a signal sequence, 2) Signal Recognition Particle(SRP) binds the signal sequence as it is translated & it also binds the Ribosome 3) There is a SRP receptor(SR) on the ER membrane which recognizes and bind the ribosome-SRP complex 4)Sec61 Translocon a water-filled pore that forms a pore through the memebrane, Sec61 translocon grabs the ribosome and its complex, and the protein is translated through Signal Recognition Particle(SRP) and Ribosomes -Polypeptides that are form in the ribosomes, exit the ribosome through a tunnel in the Large subunit(60S) -Amino acids surrounding the tunnel are neutral or polar, -The tunnel is too small for Tertiary folding polypeptides are unfolded as they come out -around the exit site, there are multiple binding site on the ribosome for the ER targeting mechanisms(e.g.SRP) -The length between the Peptidyltransferase site where the protein is made, and the exit site is 30-40 amino acids in length -Made of 6 proteins subunits and 1 RNA that forms a scaffold for the subunits so SRP is a Ribonucleoprotein -1 subunit recognizes signal sequences -1 subunit is a GTPase[binds and hydrolyzes GTP] -1 subunit is on the other end of the RNA molecule and this subunit regulates translation of the ribosome RNA strand Is bound by all the subunits at different regions of it, -This RNA strand is flexible and links everything Flexible linker -SRP checks out ALL the polypeptides as they come out of the ribosome because it doesn't know before hand if the protein coming out will have a signal or not… -when signal sequence comes out, SRP will recognize the signal and SRP will bind onto the Ribosome -The flexible RNA scaffold of the SRP will bend and bring its Translation regulatory subunit to reach into the small and large ribosome subunits and interact with the peptidyl-transferase site and pause the translation -AT THE SAME TIME, when SRP recognizes signal sequence, SRP changes conformation so that it can bind GTP -Now SR comes into play… SRP Receptor is a transmembrane GTPase -In GTP-bound state, the SR binds to Ribosome-SRP complex which is ALSO in GTP-bound state The Ribosome that is bound to the SRP-SR complex will move itself seal its exit site onto the Sec61 Translocon water-filled pore of the ER membrane that is located right next to the SR -All four shit are tightly bound to eachother until GTP hydrolysis by both SRP and SR which causes SR and SRP to dissociate from the Ribosome-Translocon complex -With the SRP(along with its Translation regulatory subunit) removed from the Ribosome, the translation is unpaused and can resume normally -With the Ribosomes exit site bound to the Translocon, the translating peptide will move through the translocon into the ER lumen as it is formed! Note: The polypeptide’s hydrophobic region do not contact the cytosol since it directly goes through the Pore The ER Translocon (Sec61 Translocon) Cluster of transmembrane alpha helices -Alpha subunit forms pore with Two parts -Beta and Gamma subunits are also present to support the pore -interior of pore is filled with water so it is Polar, so it will not react with any of the hydrophobic sequence of the signal protein. -When the pore is inactive where it is not in use, thus with no ribosome on it, it will be closed by a plug -When the pore is active, it will open but since it is tightly sealed onto the ribosome, the ionic compositions will not be able to move from the cytosol side to the ER side. -insertion of signal protein will cause the translocon to open -Note: this translocon can ALSO open laterally, where the two parts of the Alpha subunit will separate a bit so that TM helix proteins can be inserted into the membrane Translocation(secretory proteins) -Signal sequence contacts the translocon and causes the plug to be pushed aside to open the pore -Polypeptides are translocated in an unfolded state! -Movement of polypeptide is driven by energy created by the peptidyl transferase reaction of the translation -Signal Peptidase enzyme will remove the signal sequence at a site depending on the pattern(not sequence specific, but pattern specific) often the signal remains as part of the membrane Integration of TM Helix -The protein translocated, as mentioned above, does nto have strong association with the membrane, -The same pathway inserts these TM proteins -Classified by two types: • Type 1: single TM helix with N-terminus in lumen/extracellularspace and C-terminus in Cytosol • Type 2: single TM helix with N-terminus in Cytosol and C Terminus is in the Lumen Type 1 TM domain with signal sequence – The Protein has Signal at the N-terminus, and a middle region that will go inside the Membrane – signal sequence starts translocation by entering the translocon – The translocon lets in the polypeptide go through until it recognizes the TM domain by its hydrophobicity, and translocon opens side-ways letting in the Domain through laterally – The whole TM helix polypeptide is being let through during translation – After process is complete, Signal Peptidase will cleave the signal, the translocon will close up. Type I Signal Anchors -Signal Anchors is a polypeptide where the signal itself becomes the TM protein -NOT CLEAVED OFF, can be in different areas within the polypeptide -The Orientation of the Polypeptide, whether it will be Type 1 or Type2(Nterminus in Cytosol or Lumen) is determined by the Polar residues(Charges) around the signal that are recognized by the translocon, -Translocon will always put the +ve charges in the Cytosol and the –ve charge in the Lumen Translocon has many rolesopen up laterally, detect TM helix, and also detect charges on either sides of the TM domain to determine which way around it should go(type I or II) Multi-pass TM proteins -Like single TM helic proteins, there are TM proteins with multiple Helices -can be combination of Signal anchors and TM domains The organizations of the TM proteins can be predicted through a polypeptides Primary sequence (i) through the total hydrophobicity(for total number of TM helices) and (ii)charges around the helices( for the orientation in membrane), and (iii) through the modification(since Disulfide bonds only found in the Lumen, and phosphorylation only found in the cytosol) Tail Anchored Proteins • Proteins with a single C-terminal(TM domain at the C-terminus) Type 2 TM(bulk of the protein should be in the cytosol), no signal sequence(role done by TM domain) Normally 30-40 amino acids inside the ribosome(between peptidyltransferase site and the Exit site) -If TM domain is 24 amino acids long, even with the charges on either side, it is still shorter than the ribosome which means that when the C-terminus is translated, the protein is no longer attached to the ribosome (TM domain only comes out after translation is finished) – SRP cannot work because it has to bind signal and ribosome So if the protein is not attached to the ribosome.. SRP cannot work • BAT3 complex also checks out every ribosomes, but doesn’t need ribosome in order to work so it can recognize TM sequence even after release from the ribosome • After BAT3 recognizes the appropriate TM sequence, BAT3 will transfer the TM sequence to TRC40 targeting complex [So all these proteins are not created by ribosomes that are already on the ER??] • TRC40 will move the protein to the ER for insertion TRC40 is a Homodimer Nucleotide state will determine how it will interact with the substrate(Targeting polypeptide) • In nucleotide-free state , TRC40 will receive the TM domain from BAT3 • In ATP-bound state, TRC40 will close onto the Tail anchor(the TM domain) AND in same ATP-bound state it will dock onto integral membrane GET1/2 receptor which is on ER membrane • When it binds GET1/2, TRC will Hydrolyze ATP which will move the Tail anchor from TRC40 into the membrane through the GET1/2 receptor • ATP hydrolysis will also allow TRC40 to dissociates from GET1/2 and recycle back to Nucleotide-free state for another cycle! SIMILAR TO SRP  N-linked Glycosylation (Asn=N=Asparagine) Secretory proteins also go through modification as they move through Glycosylation occurs on the –NH2 of the Asparagine residue’s side chain when it is bound as followed: Asn-X-Ser/Thr motif where there is an -Asparagine linked to -X(any amino acid) then linked to either Serine or Threonine -ONLY ONLY ONLY Asparagine… Glutamine’s(Gln) amide is not even recognized -The glycosyltion is always made of 2x N-acetylglucosamine , 9x Mannose, 3x Glucose All 3 types are sugars is a branched mannose polymer with 3 glucose residues at the end -Glycans that are added through glycosylation can be modified after addition, but are never removed unless protein is degraded -Since the protein is unfolded as it translocates out the ribosome, all the Asn in the primary sequence is exposed, thus ALMOST ALL of the Asn-X-Ser/Thr motifs are going to be glycosylated • Most secretory proteins have this oligosaccharides (glycans) covalently attached which serves to: – help stabilize the native state – protect against proteases – function in cell surface signaling Glycosylation Process The Oligosaccharides are always synthesized in their attached formattached to Dolichol phosphate, which is a specialized phospholipid in the ER membrane -The enzyme that transfers this Oligosaccharide is Oligosaccharyl Transferase which attaches to the Translocon and reads what is coming out, and glycosylates accordingly during translocation -The same mannose-rich glycan is added for glycosylation -The glycan also functions to signal in the ER in order to get the protein to its folded state (ER quality control) • Glycan is modified in Golgi after exit from ER ER Chaperone System • BiP (like HSP70.. same cycle and shit!!!) – ERdj proteins (DNAJ co-chaperones) – NEFs • Grp94 (HSP90) – no co-chaperones • thioredoxin family – PDI and ERp57 • calnexin / calreticulin • UGGT (UDP-glucose:glycoprotein glycotransferase) • glucosidases, mannosidases, lectins (glycan binding) • Degradation takes place on cytosolic proteasomes • Folding is necessary to exit ER to the secretory pathway Folding Quality control of Proteins can occur in cytosol or lumen Degradation Quality control of proteins ONLY OCCURS IN THE CYTOSOL cus proteasome is only in cytosol So there are proteins that recognize misfolded shit in the lumen, and remove them from ER and put em out in the cytosol so that they are degraded BiP and Co-chaperones(ERdj = DNAJ) TM and lumenal ERdj proteins activate BiP to hydrolyze ATP and shit like DNAJs: - ERdj3 is the main Type 1 DNAJ which assists in folding -Keep misfolded proteins soluble so that they don't aggregate and can be easily put thru degradation Sec63 is bound to the translocon and is a Type 3 DNAJ that recruits BiP to the translocating polypeptides [recall Type3 have J-domains which can activate HSP70, but here activates BiP to bind to polypeptide as they come through the translocon, so folding can begin during translation and translocation.] -folding starts during translocation -Also Like in the cytosol, NEFs reset BiP ATPase cycle Protein Disulfide Isomerase Cytosol is reducing environment and ER lumen is an oxidizing environment, The ER’s oxidizing environment will favor the formation of disulfide formation but is slow however PDI will catalyze and speed up the formation –PDI has 2 sites for the 2 Cys side chains of the polypeptide which bring the Cys close together for oxidation Disulfide Isomerization • Oxidized PDI becomes reduced when it catalyzes formation of disulfide bonds in substrate[making the substrate now oxidized PDI becomes reduced and substrate is oxidized • Another Major function of PDI is when PDI is Reduced, it rearranges the disulfide bonds during folding because intermediates are formed during the folding process, and sometimes the Disulfide bondsis formed in the wrong place and prevent the intermediate from reaching native state, so the Reduced PDI helps rearranges the disulfide bonds until the proper folding state is achieved. –Native disulfides are the most stable -During rearrangement, the Reduced PDI forms a disulfide WITH the substrate (the disulfide was normally on cys side chains that are within the polypeptide itself) • In both reactions,when taking disulfide, and giving the disulfide.. PDI forms mixed disulfide intermediates with substrate PDI Regeneration As all the PDI keep get reduced by forming disulfide bonds on the substrate, all the PDI can end up reduced, however there is a chemical cascade that regenerates oxidized PDI • PDI is oxidized by Ero1 protein (oxidoreductin) wihich has a cofactor called FAD • Ero1 has its own disulfide bonds that are oxidized, • Ero1 gets reduced and makes PDI oxidized • Ero1 is regenerated by cofactor • fresh FAD is supplied from cytosol to maintain oxidation potential for PDI levels to remain oxidized FAD • Flavine Adenine Dinucleotide • Carrier molecule with redox active N(nitrogen) sites which can get oxidized and reduced… unlike Ero1 it does not have its own disulfide bonds but rather an oxidized Nitrogens • Reduced FADH pr2duced in ER by oxidation to form disulfide • Reduced FADH pr2duced in mitochondria by oxidation of acetyl-CoA • [FADH2 is the same as Acetyl-CoA except instead of reactive sulfhydryl you have flavine] There are also other mechs that take reduced FADH2 and put it in Oxidized state Calnexin and Calreticulin [OTHER ER-specific chaperones] • Calnexin (CNX) and Calreticulin (CRT) are closely related.. they have same lumenal domain[~50 kDa] except CNX has a TM anchor which keeps it on the ER • CRT has a signal instead for retention in the ER • Lumenal domains recognize glycosylation pattern on polypeptides, • Lumenal domains also have another site which binds to ERp57 (is like PDI, since it also has redox ability that rearranges disulfides) • CNX has glycan binding domain(AKA Lectin) , ERp57 site , and TM anchor • Calnexin Binding • N-linked oligosaccharide(from glycosylation) is a branchd mannose polymr wit 3 glucose residues at the end • The glucose and then mannose are trimmed off step by step[first the glucoses at the end, then the mannose] • Glucosidase and Mannosidase remove the glucose and mannosidases • CNX specifically binds the oligosaccharide with 1 glucose –because CNX is attachd to the ER, it will keep the polypeptide in the ER to help its folding, –CNX prevents polypeptide exit to Golgi –Therefore single glucose is a signal for incomplete folding, which makes the protein remain in the ER until it is folded properly… but is eventually the last glucose is removed, and CNX doesn't recognize it anymore UGGT • UDP-glucose Glycoprotein Glucosyltransferase *ONLY PRESENT IN ER LUMEN* • UGGT[^] Binds non-native polypeptides that have no attached glucose, and reattaches a glucose to the oligosaccharide This allows the CNX to bind again • Glucosidase removes Glucose from BOTH native and non-native polypeptides, so it always removed it • Native polypeptides are not recognized by UGGT… • so when glucose is removed from a native polypeptide by Glucosidase, UGGT will not recognize it and not put back a glucose, leaving the polypeptide to leave freely unbound by CNX • When glucose is removed off a non-native polypeptide by Glucosidase, UGGT will recognize it and put back the Glucose everytime it is removed so that it can be bound to CNX and not leave the ER • Mannosidase trims the sugar even further, removing the mannoses and preventing the glycosylation on the residue to be evr bound by CNX agan –UGGT does not recognize shortened glycosylation which have had the mannoses removed[so the glucose cannot be attached, since the mannose cannot be attached either for UGGT to recognize the glycosylation] After Mannosidase takes action, the protein is either irreversibly misfolded, or is ready to leave Calnexin Cycle • The binding of CNX keeps the polypeptide in the ER for it to be folded by chaperones (ERp57, PDI, BiP) • UGGT restores the glucose so that CNX can bind to the single Glucose that is on the misfolded polypeptides • UGGT does not restore glucose on Folded Native polypeptides and causes them to exit to Golgi • Mannosidases trims glycans irreversibly whether the peptide is native or non-native!!! But Mannosidase is slow so the peptides can go through the Calnexin cycle many times before Mannoside can act on the protein • unfolded proteins that cycle too long in the ER are more likely to be trimmed by mannosidase • Mannose-binding lectins select proteins with short mannose-tree glycan to be sent out to the cytosol for degradation by proteasomes • ER Associated Degradation • ERAD degrades bot
More Less

Related notes for ANAT 212

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit