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Week 4 Class Notes (9/15/16-9/20/16)

by: Luke Holden

Week 4 Class Notes (9/15/16-9/20/16) BIOL 4610

Marketplace > Clemson University > BIOL 4610 > Week 4 Class Notes 9 15 16 9 20 16
Luke Holden

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These notes are detailed and contain explanations for some of the concepts. Includes lots of pictures and diagrams to help!
Cell Biology
Susan Chapman
Class Notes
endoplasmic reticulum, golgi apparatus
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This 15 page Class Notes was uploaded by Luke Holden on Wednesday September 14, 2016. The Class Notes belongs to BIOL 4610 at Clemson University taught by Susan Chapman in Fall 2016. Since its upload, it has received 25 views.


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Date Created: 09/14/16
Week 4 Class Notes (9/15/16-9/20/16) Resident ER Proteins Assist in the Translocation and Folding of Proteins Big Picture: This is the second step in the secretory pathway! Overview:  BiP- Binding Proteins  Protein Disulfide Isomerase  Glycosytltransferases o N-Linked o O-Linked BiP- Binding Proteins (Member of the Hsp 70) Heat Shock Protiens (Hsp70)  These are an abundant chaperon proteins in the ER lumen that assist with the folding of cells  More specifically the are called BiP- Binding Proteins  These guys bind to the hydrophobic regions on the nacent protein that is being cotranslated into the ER lumen o This helps bring the protein in as well as keep it from aggregating with other proteins o It is very important because thousands of proteins are being pushed into the ER and if they all stuck together then they all would be unable to work.  Important Steps: o When the protein is pushed into the cell, ATP is HYDROLYZED and BiP clamps down on to the protein o When BiP wants to release, it simply re-phosphorylates converting ADP to ATP and the protein is released  This classifies it as an ATPase o As a way of quality control (to make sure the protein folded correctly) if the hydrophobic binding region is on the inside of the cell then the protein is correct o If the hydrophobic region is on the outside then BiP rebinds and it tries again Protein Disulfide Isomerase (PDI) Important Things to know about PDI  Disuldife bonds are found on cysteine molecules  This molecule helps with structure and folding of a protein  Soluble  Sulfur is crucial  You want your bond to look like this:   How PDI Works o IT IS ESSENTIAL THAT YOU KNOW REDOX REACTIONS LIKE THE BACK OF YOUR HAND!! o “THIS WILL BE AN EXAM QUESTION” o OIL RIG: 1. Oxidation involves loss (or having loss) of a proton 2. Reduction involves gain (or having gain) of a proton 3. o Steps For FORMING A DISULFIDE BOND 1. Reduced protein comes along to meet up with an oxidized PDI 2. PDI is reduced (gets the proteins electrons) as the sulfur on the Cys gets oxidized 3. This positive charge (electrophile) is attached by the next sulfur (nucleophile) on the protein 4. The second sulfer is then oxidized giving the proton to PDI 5. Now you have a DI sulfide bond 6. PDI is then oxidized by ERO1 (which ERO 1 is reduced) now PDI can return and help for another disulfide bond. o STEPS FOR REDUCING DISULFIDE BONDS 1. We don’t know how this happens but we thing it is because PDI can enter the hole created by the misfolded protein and a correctly folded protein does not leave a hole 2. PDI now starts REDUCED 3. it comes and donates (oxidized) electrons to the misbonded disulfide thus the reaction is reversed 4. Then the process can resume as normal What happens if the misfiled protein can’t be fixed?  all proteins want to be stable  They would rather die (in no way are they living but just metaphorically speaking) than be unstable  Unfolded protein Response o ERAD (ER Associated Degradation)  Proteasomes come and degrade protein  sugar molecules are crucial for this recognition Protein Routing is controlled by many factors:  many proteins synthesized in the ER are glycoproteins which are tissue specific  insital glycosylation occurs as soon as the protein enters  the golgi complex helps modify protein  KDEL (Lys-Asp-Glu-Leu) o These are for proteins whose final destination is the ER o The Golgi complex has a receptor protein that binds the KDEL sequence and delivers the protein back to the ER ON HER SLIDESHOW FOR “PRESENTAITON SLIDE SET 4- ENDOMEMBRANES II” STOP HERE!!! YOU DON’T HAVE TO KNOW ANY OF THEE MATERIAL BEYOND THIS POINT OF THIS POWERPOINT BEGIN WITH SLIDE 30 ON “Slide Set 5- protein targeting and sorting” (we will cover mitochondrion but just to keeps thing consistent with the ER we are starting here). Glycosyltransferases Glycosylation- the addition of carbohydrate side chains to proteins Glycoproteins are made here and come with a modified oligosaccharide side chain N-Linked O-Linked Nitrogen-Linked Oxygen-Linked Addition of and oligosaccharide to Addition of and oligosaccharide Serine Asparginine or threonine N-Linked:  Refer to slide 33 This is the big picture on where the glycosylated protein goes On the Next page we will talk about how to make this   We want to make this: two units of N-acetylglucosamine, nine mannose units, and three glucose units   Steps: o Glycosylation begins as dolichol phosphate, an oligosaccharide carrier, is inserted into the ER membrane o GlcNAc and mannose groups are then added to the phosphate group o The growing core oligosaccharide is translocated to the ER lumen by a flippase o Once inside the lumen, more mannose and glucose are added o The completed core oligosaccharide is transferred from dolichol to the asparagine residue of the recipient protein o The core oligosaccharide attached to the protein is trimmed and modified o Once it has reached the end of the line it is transferred all together (en massae) to the protein o From there is under goes quality to control check before being exported to the golgi:  9/20/16 Lecture The glucoses are taken off one by one until it reaches the end. The If all is well a mannose is removed and it is exported to the golgi Unfolded Protein If not all is well then a glucose will reattach to the mannose and it will stay in ER Response for a long time  If not fixed than it is destroyed (coming up next) So: 321 are we good?  Yes  Mannose GO (2Glc and 8 Mann) 321 are we good?--> No  Glucose and sit (2Glc 9 mann I Glucose) To remember this process just think pf Dr. Chapman saying Chop, Chop, Chop. Remember, these are the proteins that get stuck in the ER for to long that cant be fixed  So, this is like a recycling process in which BiP is brought back because BiP is so important.  It does this by: 1. Binding to the BiP proteins in the ER Lumen which were original attached to the 2 monomers Ire1. 2. The release of BiP from the monomer casue the two Ire1 monomers to form a dimer which activates ints endonuclease activity 3. Next, the pre spliced Hac1 mRNA comes and the intron in spliced out by the Ire1 dimer 4. The mature mRNA is then translated and the resulting Hac1 protein returns to the nucleus where it catalyzes the transcription of BiP ( a protein folding catalyst) Assisting in protein folding  2 proteins assist in protein folding o calnexin (CNX) and calreticulin (CRT) (they are both lectins- sugar molecule attachers) o These bind to monoglucosylated glycoproteins and assist with protein folding o This occurs in a complex that includes thiol reductase know as ERp57 o The complex is removed and the final glucose is removed by glucosidease II o Steps 1. glucosyl transferase, UGGT, (UDP-glucose:glycoprotein glucotransferase), binds to improperly folded proteins 2. It adds back a single glucose unit, making the protein a substrate for CNX/CRT binding 3. Once proper conformation is achieved, UGGT no longer binds the new glycoprotein, which moves on to the Golgi Call glycosyltranferase (puts a glucose back on) After removing the glucose, if we have no hope of folding, it will undergo EDEM degradation or OS-9 degradation. Recognition by either EDEM or OS-9 leads to dislocation of the misfolded protein out of the ER, ubiquitination, and degradation by the proteasome. calnexin (CNX) and calreticulin (CRT) come and bind and try to rearrange the protein by promoting disulfide bonding ER protein is in normal process and OOPS THE PROTEIN IS NOT FOLDED CORRECTLY Mitochondrion and Protein Targeting Facts: o you get your mito from your mother o 57 known genes in the mito o mito obviouslt need more than this o gets extra genes from the nucleus o so its kind of like a we need you but you need us relationship  we need the mito power  they need the nucleus DNA o mito encoded : o mito- encoded are transcribed in the mito (THIS IS NOT A MISTAKE! PLEASE KNOW THAT THIS IS A SPECIAL KIND OF TRANSCRIPTION BECAUSE IT TAKES PLACE IN THE MITO o mito- encoded translated on its OWN ribosomes in the intracellular ribosomes o other proteins are also transcribed and translated in the mito o Nuclear Encoded o The nuclear encoded proteins are just like the old stuff you know where it is transcribed in the nucleus and then translated in the cytosol o then it is imported into the mitochondrion o Importing proteins into the mito o Requires 4 protein/ protein complexes  Import Receptor  reads mitochondrial targeting signal  delivers to translocon  Translocon of Outer Membrane (TOMs)  associates with import receptor  delivers protein to second translocon  Translocon of Inner Membrane (TIMs)  aligned with TOM40  Matrix Hsc70 (acts like BiP)  aids in net translocation o Targeting sequences for the mito (starting to notice a pattern here?)  this sequence is hydrophobic, basic, and hydroxylated residues  and is cleaved once it enters the mito  N-terminus located  20-25 AA long  forms and amphipathic helix o Most of the proteins are folded in the cytosol o Hsc 70 proteins come and bind to the newly synthesized protein and hydrolyze ATP to keep the protein linear (like a shoe string) o Bringing in the protein to the matrix on the next page! Step 1: The import receptor binds the protein and prepares it for entry Step 2: TOM and TIM line up creating a hole directly to the matrix the space (don’t have to know the names of tom and tim but know that TOM 40 is the main translocon of the outer membrane) note that if a protein is just going to the IMS then it skips TIM! Step 3 Upon entry of the matrix the targeting sequence is then cleaved off by the matrix processing protease and new Hsc 70 (matrix) proteins bind to keep that protein straight Step 4: The protein is finally translated HIGHLY BASIC IN THE MATRIX THIS FURTHER INTIATES PROTEIN FOLDING o Applying proteins to the mitochondrial membrane is a different story: o The orientation HAS TO BE CORRECT!!!! o 3 path ways to a mitochondrial membrane o Chart on next page Target Example Steps Picture Sequence Path Hydrophob Cytochrom Import A ic Stop- e oxidase receptor transfer subunit TOM 40 sequences TIM Complex Signal Cleavege Released by TIM into IM Path Internal STP TOM40 B sequences synthase TIM recognized subunit 9 Signal by Oxa 1 Cleavage Released into matrix Oxa1 inserts into IM Path Mulitple ADP/ADP Import C sequences antiporter receptor- recognized (TOM70/TOM by TOM 70 20) and TIM 22 TOM 40 TIM(22/54) released by TIM into inner membrane and facilitated by TIM9 and 10 Golgi Apparatus o ER and glogi are almost continuous o Structure and function of golgi: Don’t have to know all the steps just important fx include: Sugar molecules are added and removed Terminal sugar is modified (this is like the golgi tool box) it can craft the sugar and make it whatever it wants it to be. o Transport Vesicles This supplies thee movement of solutes through the golgi o 2 Models o The Stationary Cisternae Model  In this model, each cisterna in the Golgi stack is a stable structure  Transport of materials from one cisterna to another is mediated by shuttle vesicles  These bud off from one cisterna and fuse with the next cisterna in a cis-to-trans sequence o The Cisternal Maturation model  In this model, each cisterna in the Golgi stack is a stable structure  Transport of materials from one cisterna to another is mediated by shuttle vesicles  These bud off from one cisterna and fuse with the next cisterna in a cis-to-trans sequence   Retrograde movement (Cop1): Backwards  Movement of protein back to ER after final modification  This allows the cell to balance the flow of lipids toward the plasma membrane  It also ensures a supply of materials for forming new vesicles  Proteins whose final destination is the ER have a KDEL sequence (Lys-Asp-Glu-Leu) or related sequence  The Golgi complex has a receptor protein that binds the KDEL sequence and delivers the protein back to the ER  Anterograde movement (Cop3): Forwards  Movement of material toward the plasma membrane is called anterograde transport  As a secretory granule fuses with the plasma membrane and discharges its contents (exocytosis), a bit of membrane from the ER becomes part of the plasma membrane  This flow of lipids toward the plasma membrane must be balanced


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