Biol 408 Exam 1 Study Guide
Biol 408 Exam 1 Study Guide Biol 408
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This 11 page Study Guide was uploaded by Amanda Tobias on Monday October 3, 2016. The Study Guide belongs to Biol 408 at Towson University taught by Dr. Elana Ehrlich in Fall 2016. Since its upload, it has received 125 views.
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Date Created: 10/03/16
Secretory pathway Proteins that go through the secretory pathway are those that are ultimately secreted, membrane proteins, resident lysosome proteins, and proteins that live in the R and Golgi as well. • microsomes and studying the secretory pathway • microsomes are formed when you disrupt the cell • Rough and smooth microsomes form and they are separated by centrifugation • Microsomes form from the disrupted ER and have the same topology and function as the ER • One experiment was done to test the signal hypothesis, which proposed that proteins destined for secretion are originally manufactured with an initial sequence of amino acids that may or may not be present in the mature protein. Invitro translation of proteins was done with and without microsomes and the protein was smaller when the microsomes were present. That showed that the protein was cleaved somewhere when it was made in the ER. • role of SRP and signal sequence in import to the ER • Proteins are brought into the ER co-translationally • SRP brings the ribosome and mRNA/protein being translated into the ER and translation is finished into the ER • New polypeptides and associated ribosomes are targeted to the ER through recognition of a signal sequence by SRP (signal recognition particle) • Signal sequence is the first 20-50 amino acids of a protein and they are a sequence that is recognized by SRP, which binds to the signal sequence • When the SRP binds to the signal sequence, it pauses translation and drags the whole complex to the ER • Translation resumes once the whole complex is in the ER and the protein is made into the ER • signal peptidase, integral membrane proteins, how to read a hydropathy plot • Signal peptidase recognizes the cleavage site and cleaves the signal sequence from the polypeptide in the ER • Integral membrane proteins are translated into the ER and move into the membrane co-translationally just like other proteins going through the secretory pathway. • While the protein is being translated into the ER there is a stop transfer sequence that determines what is going to be the transmembrane domain. The protein will stop translating, embed itself into the membrane and then continue translating. The protein will slide out of a translocon and into the membrane. Then the translocon gate closes. • The cytosolic side of the protein will stay the cytosolic side. The part of the protein that is in the lumen of the ER will eventually be outside the cell. The protein is transported in the membrane of a vesicle to the plasma membrane. • An internal signal sequence can be used to integrate a single pass transmembrane protein into the ER membrane in two different ways: • The same sequence can act as a stop transfer and signal sequence. This sequence is not cleaved • The N terminis is always translated first and C terminis is translated second • Hydropathy plots are a measure of a protein’s hydrophobiticy • The 20-30 amino acids that span the plasma membrane must be hydrophobic. • When reading a hydropathy plot, if there is a region that is broad and high above 0 on the hydrophobic side of the plot, it is probably an integral membrane. By counting the amount of peaks, you can tell how many times it passes through the membrane. what happens in the ER • Protein folding (chaperones, disulfide bonds, PDI, PPI, glycosylation, role of glycosylation in protein folding, assembly into multisubunit proteins) • Disulfide bonds are formed and rearranged • They stabilize tertiary and quaternary protein structure • Disulfide bonds for between adjacent cystines but sometimes that isn’t where you want them to form, so they need to be rearranged • PDI • Protein disulfide isomerase (PDI) catalyzes rearrangement of disulfide bonds in order to have the correct bonds and form the correct structure • PDI is an ER resident protein • PPI • Peptidyl-propyl isomerases (PPI) are enzymes that rotate amino acids around the peptide bond in order for the protein to fold properly • Chaperones • General term for proteins that assist other proteins in folding correctly • Glycosylation • An oligosaccharide is transferred as a unit to the protein • Oligosaccharyl transferase active site is in the lumen of the ER. If transfers the big sugar (oligosaccharide) to the growing polypeptide chain in the ER • Aspargine (N) liked oligosaccharides are added to most proteins in the ER • N-linked oligosaccharides serve as a binding site for calnexin and calreticulum on proteins and together they retain the protein in the ER until it is properly folded • Folding cycle and calnexin binding is regulated by glycan code • Calnexin holds on to the protein until it folds. Then glucosidase cleaves calnexin off. Fi the protein is folded properly it can leave the ER to go to the Golgi. If it isn’t, glucosyl transferase binds and adds another glucose so it can bind again to calnexin. This repeats until it is folded correctly • quality control (ERAD, UPR) • Both ERAD and UPR are used when there is a build up of misfolded proteins in the ER • ERAD • ER associated degradation • Irreversibly misfolded proteins are retrotranslocated, ubiquitinated, and degraded by the proteasome • Ubiquitin • 76 amino acid protein tag • covalently conjugated to other proteins • present in all Eukaryotes • conjugated to lysine • One of its function is targeting proteins for degradation via the proteasome • Substrate + ubiquitin = ubiquitin conjugation • Ubiquitin is added to the protein covalently in 3 steps, E1, E2, and E3 • Enzymes will remove polyubiquitin chain to be recycled and the protein will be degraded • UPR • Unfolded protein response • IRE1 • Example of a sensor of misfolded proteins that activates gene expression • Misfolded proteins in ER signal the need for more ER chaperones by activating a transmembrane kinase • Activated kinase turns into an endoribonuclease • Endoribonuclease cuts specific RNA molecules at two positions, removing an intron • 2 exons are ligated for form an active mRNA • mRNA is translated to make a gene regulatory protein • Gene regulatory protein enters nucleus and activates genes encoding ER chaperones • Chaperones are made in ER, where they help fold proteins • A sensor of unfolded proteins in the ER induces the expression fo genes involved in ER expansion and protein folding Moving out of the ER to the Golgi • vesicular transport (Rab and SNARES) • properly folded proteins exit from the ER and travel to the Golgi for further maturation • Proteins move between compartments in the secretory pathway by means of vesicular transport • Proteins are always in vesicle, never in cytoplasm • Proteins move out of the ER in vesicles whose contents are transported through homotypic or heterotypic fusion, forming vesicular tubular clusters • ER derived vesicles fuse to form vesicular tubular clusters, packages of proteins that move along mictotubules from the ER to the Golgi) • Transport vesicles have protein coats that function in vesicle formation • Protein coats help to form the curvature of the membrane • Examples are clathrin, COPI, and COPII • The vesicle coat concentrates certain membrane proteins in a region that will give rise to the vesicle. • Assembly into curved basket type lattice promotes shape • Once you release from the membrane, there is dissociation of the coat • SNARES • There are SNARES on the vesicle and the target membrane • V SNARES are on the vesicle • T SNARES are on the target cell • Both SNARES wrap around each other to exclude water and fuse the membranes • SNARES must dissociate so they can be recycled and participate in another round of vesicle transport • SNARES are membrane proteins • When fusion occurs the vesicle membrane becomes part of the Golgi membrane • Rab • Different Rab proteins are in different compartments of the secretory pathway and help determine specificity of transport • Rab proteins facilitate docking and SNARE pairing • Different Rab proteins and effectors and in different organelles, which help with the specificity of vesicular transport • Their common function is tethering vesicles to appropriate membranes and triggering the release of SNARE control proteins • There are 760 Rabs that mediate transport of proteins in vesicles between different membrane • Concept of GTP binding proteins • GTP binding proteins are molecular switches • They regulate many processes and activity is controlled by GTP/GDP bound state • GTP binding proteins are controlled by GAPs and GEFs • The protein is off when it is GDP bound. GEF comes and helps release GDP so GTP can bind • When GTP is bound, it is turned on GAPs help release GTP to turn off the gene again • Conformational changes are caused by GTP hydrolysis • Rab proteins, and GTPases help to determine the specificity of vesicular targeting • Rab-GDP is activated by Rab GEFs on the membrane that bind Rab and interact with the t-SNARE to fuse with the membrane ER retrieval pathway Sometimes ER resident proteins end up in the Golgi and there needs to be a way to retrieve them KDEL receptors bind to all the proteins of KDEL in the acidic Golgi. They get put in vesicles to go back to the ER. They don’t bind well in the ER because of the more neutral environment There is a pH gradient KDEL is the ER retrieval signal pH gradient allows KDEL to bind in the Golgi and release in the ER ER resident proteins tend to stick together and form aggregates that make it hard to leave the ER in the first place What happens in Golgi? Proteins and lipids enter the cis Golgi network from the ER and exit from the trans Golgi network to lysosomes, plasma membrane, or secretory vesicles • Trimming and modification of sugars • N-linked oligosaccharides are trimmed • In lab you can use Endo H and by determining if a protein is endo H sensitive or endo H resistant you can tell if it is in the early or late secretory pathway • Trimming of N linked oligosaccharides • O-linked glycosylation (added to OH of serine and threonine) • Proteoglycans • Sulfation of glycosaminoglycan • Benefits of Glycosylation • Protein Folding • They protrude from the protein’s surface and limit interactions with other proteins such as protease • Cell adhesion • Regulation of cell signaling • addition of M6P to acid hydrolases • Lysosomal proteins are tagged with mannose 6 phosphate • it is added to the N-linked oligosaccharide in the Golgi • There is a M6P receptor in the Golgi that binds to all proteins that are being tagged with M6P and the vesicle that goes to the lysosome is formed • A signal patch in the lysosomal hydrolase is recognized by the GlcNAc phosphatransferase that will add the M6P tag in the Golgi • The signal patch is a bunch of amino acids that when folded are close to each other • The signal patch binds to the recognition site, transfer of GlcNAc phosphate to mannose in the catalytic site happens, and acid hydrolase with GlcNAc-phosphate is attached to mannose in the oligosaccharide • What are acid hydrolases • Proteins that live and work in the lysosomes • They help degrade the proteins that go to the lysosome to be degraded Lysosome • how things get there – things that are degraded vs. things that are doing the degrading (example :acid hydrolases), how M6P is added, what is M6P, how are acid hydrolases with M6P transported from the Golgi to lysosome • Acid Hydrolases • M6P modified acid hydrolases are bound by the M6P receptor in the Golgi • M6P receptor shuttles back and forth between the Golgi and late endosomal membrane • Mechanism for binding lysosomal proteins in the Golgi and releasing them in the late endosome • M6P picks up in the Golgi and dropped off in the endosome. It is regulated by ph. pH in the Golgi is about 6.6, which is the idea pH for M6P to bind to the receptor. The pH in the early endosome goes down to 6. • Retromer coat surrounds the vesicle with M6P receptor to bring it back to the Golgi • There is a clathrin coat around the forming vesicle and a Rab that helps the vesicle get to the lysosome • Phosphates in the lysosome remove M6P and prevent it from rebinding to the M6P receptor and going back to the Golgi • Sometimes acid hydrolases accidently get secreted. There are M6P receptor that go to the membrane and bind to the acid hydrolases and bring them back in • Things that are being Degraded • Get to the lysosome by endocytosis • Go from cell surface inward to the lysosome • Phagocytosis by specialized cells • Pseudopod formation driven by actin polymerization and reorganization • Clathrin coat is used to form vesicles in endocytosis • Unless cargo is diverted elsewhere, the default pathway for molecules taken in through endocytosis is the lysosome • Cargo is brought in through coated vesicles, and goes from the plasma membrane to the early endosome, late endosome, and then lysosome • Regulation • The phosphatases in the lysosome remove M6P to prevent it from rebinding to the M6P receptor and going back to the Golgi. • If and acid hydrolase does make it to the plasma membrane to be secreted instead of going to the lysosome, there are M6P receptors on the plasma membrane to bind the acid hydrolases and bring them back in. Endocytosis, receptor mediated endocytosis From the cell surface inward to the lysosome General Endocytosis: A signal sequence determines recruitment of a plasma membrane protein into clathrin coated pits for endocytosis Specific sequence in the cytoplasmic tail of the receptor that is involved in the recruitment of the protein into clathrin coated pits It is involved in the interaction with adaptin It mediated interaction with adaptin An endocytosis signal = Y-X-X- ψ X= any polar amino acid Ψ = any hydrophobic amino acid LDL receptor is an exception Many different receptors can accumulate in a clathrin coated pit because they all have some form of the same nonspecific signal sequence Phagocytosis By specialized cells Pseudopod formation driven by actin polymerization and reorganization Clathrin coat is used to form vesicles in endocytosis Caveolae also form pinocytic vesicles Caveolae aren’t proteins. They are just a form of pinocytic vesicles that are made up of regions of membrane that are enriched with lipids, cholesterol and proteins Invaginate based on lipid composition of membrane rather than protein Viruses enter through caveolae • Different examples of recycling and for receptor down regulation • LDL Receptor • Example of recycling the receptor • LDL binds to receptor, goes to early endosome, recycling endosome, the receptor dissociates. The LDL goes to the maturation endosome and then the lysosome and the receptor goes to recycling endosomes • pH causes the Ligand to dissociate from the receptor and the ligand to go to the lysosome and recycling endosome for the receptor • Transferrin Receptor • Example of recycling the receptor and ligand • Ferrotransferrin is iron bound • Apotransferrin has no iron • Iron is released upon acidification of late endosome. Transferrin and transferrin receptor remain bound • Transferrin receptor plus apotransferris us recycled to the plasma membrane • This is an economical way for the cell to take up iron because everything is recycled so you don’t need to make more. • This is different from LDL because LDL gets degraded and in this case it gets recycled • This is an easy way to down regulate because an easy way to turn things off is to degrade them • Regulation of receptor down regulation by ubiquitination and sequestration/degradation via MVB • Receptors can also be down regulated through sequestration in multivesicular bodies (MVB) • By using MVBs, the cytoplasmic tail can’t interact with proteins in the cytosol because it is hidden in the MVB, so signaling can’t happen • Recruitments of receptors into clathrin coated pits is regulated by mono or multiubiquitination of receptor cytoplasmic tails • Ubiquitin binding proteins recruit receptors into clathrin coated pits and later recruit ESCRT complexes to sort receptors into the ILV (intraluminal vesicles) • Ubiquination to MVB to lysosome is a way to shut off signaling. It also recruits specific receptors for this process. • Receptors are brought into the cell via endocytosis and then bud into the endosome to form a MVB • A signaling receptor with ligand and ubiquitin tag attached is invaginated and pinched off (sequestration) and that becomes a MVB that fuses with the lysosome • This formation required an ESCRT complex Exocytosis • Constitutive • Happens all the time and is unregulated • This is the default pathway for all proteins leaving the Golgi unless they are diverted to: • Signal mediated diversion to lysosomes • Constitutive secretory pathway • Signal mediated diversion to secretory vesicles for regulated secretion • Back to ER as ER resident proteins • Regulated secretion • Controlled. The vesicle won’t form with the plasma membrane until there is a signal • Secretory vesicles contain specialized products that are secreted in response to a signal • Proteins get packaged into secretory vesicles and just wait there until they get a signal to be released. Nuclear Import Active transport Proteins that contain a nuclear localization signal (NLS) localize to the nucleus They are short sequences enriched with lysine and arginine Mutating the amino acids in the NLS stops it from going into the nucleus Nuclear import receptors bind to the NLS in cargo protein as well as F-G repeats on nucleoporins They Facilitate nuclear import then return to the cytoplasm It requires a GTP depended Ran Ran-GTP binds nuclear import receptor (importin) in the nucleus, stimulating cargo release, brings importin back out to cytoplasm Ran-GTP is in the nucleus. It binds to the nuclear import receptor and releases the cargo in the nucleus. It then goes back to the cytoplasm when GTP is hydrolyzed to GDP and releases the importin it brings out with it Directionality of nuclear import: Ran-GAP stimulates GTP hydrolysis in the cytosol and Ran –GEF restores Ran-GTP in the nucleus Ran-GAP is localized in the cytosol, so Ran-GDP is in the cytosol Ran-GEF is localized in the nucleus, so Ran-GTP is in the nucleus Binding of import receptor by Ran-GTP alters NLS recognition site, forcing release of cargo Common mechanism: Nuclear localization can be regulated by controlling the visibility of the NLS to the nuclear import receptor High Calcium concentration when T cell is activated Activates calcineurin Calcineurin binds to protein and exposes the nuclear localization signal Protein goes in the nucleus when there is low calcium concentrations so calcineurin releases The nuclear import signal is hidden then and nuclear export signal is exposed Protein is released Nuclear Export Nuclear export requires a nuclear export signal (NES) and receptor Similar mechanism to nuclear import Ran-GTP mediates interaction between exportin and protein to be exported. They all bind together and exit the nucleus When Ran-GDP is hydrolyzed it releases the exported protein Ran regulates the biding and/or release of cargo from the export/import receptor GTP hydrolysis promotes release of import receptor in import GTP hydrolysis promotes release cargo in export
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