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Lecture 4

Biology 2290F/G Lecture 4: Protein Synthesis and Transport & Endoplasmic Reticulum

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Biology 2290F/G
Ray Zabulionis

Lecture 4: Protein Synthesis and Transport + Endoplasmic Reticulum Protein Sorting/Targeting - Typical mammalian cell: 10 000 proteins, must be localized correctly - Newly made peptides must be directed to the correct destination - Targeting: o Direct proteins to the right destinations (organelles) o During or after synthesis - Sorting: o Direct proteins to the secretory pathway (ER, Golgi, lysosomes) - Proteins have to get to the right destination - How do they do that? Targeting - We have a targeting signal, which is like your address that tells the mailman to deliver to your address o Targeting can occur during/after synthesis - Targeting generally involved with signal sequence that tells a cell to take a protein somewhere, usually directing it to an organelle - Sorting relates more to directing proteins to the secretory pathway o Get the cell to the ER, and then vesicles will be directed towards different pathways in/out of the cell General principles of protein synthesis, targeting and sorting - Many proteins are synthesized just by cytosolic ribosomes: o Those which remain in the cytosol o Those which are targeted to intracellular organelles such as (ER), mitochondria, chloroplasts, peroxisomes, and nucleus (they have a specific signal sequence) - Other proteins are synthesized by ribosomes attached to ER (the rough ER) o Those which reside in the ER and proteins which are sorted to PM, Golgi complex, and lysosomes - Accordingly, two major protein-sorting pathways are known: nonsecretory and secretory Basic mechanisms of protein targeting to the membranes are common - Signal sequence - Receptor for the signal sequence - Translocation channel - Source of energy - Protein is completely made in the cytosol, has protein sequence that can direct it to other organelles within the cell - We have other proteins that start to be made in the cytoplasm, but they're destined for the ER - Many proteins destined for secretion ER Structure - Uninterrupted membranous tubules and vesicles separated from cytoplasm - RER has ribosomes on the tubules (cisterna) - Cisterna are stacked - ER extend from nuclear membrane - ER is an extension of the nuclear membrane - Tubular structure - Exam question: what microscopy produced this picture? Stringing the Concepts Together - Identify cellular features by microscopy, isolate and homogenize them to free organelles - Sucrose density-gradient centrifugation of homogenate allows for isolation of microsomes and ribosomes - SDS-PAGE is used to identify newly translated proteins Secretory proteins enter the ER lumen - Experiment showing that secreted proteins must first go through the ER - How do proteins get from the inside of cells to the outside? - Microsomes have ribosomes attached to them o We want to know if there are proteins inside of them - People took isolated microsomes and then split it into 2 o One sample was treated with detergent, punctured holes in the membrane, then added a protease o Other sample did not get detergent, but protease was still added - Two samples were then analyzed with SDS-page - The idea was to determine if these proteins were found within the lumen of the ER o If that was true, without a detergent, the proteins would be protected o If we added protease after detergent treatment, the protease could get inside and chew up all the proteins - Proteins destined for secretion have to go through the ER and enter through the lumen ER Functions - Secreted and membrane proteins are sorted through RER - Sugars/carbohydrates are added to the polypeptide - Disulfide bonds are formed - Proteins are folded by “chaperones” Translocation and translation occur simultaneously - Cell-free experiments demonstrated that translocation of secretory proteins into microsomes is coupled to translation - Is a protein made in the ER? - Scientists did an in vitro translation o Took a concoction of proteins and synthetic RNA and they mix it all together - Mix this with microsomes and see how many proteins we can find in there - Microsome removes the signal sequence RER: What are the Major Players? 1. Amino terminal signal sequence of newly initiated polypeptide (nascent proteins) 2. Signal-Recognition Particle (SRP) 3. SRP receptor embedded in ER membrane 4. Translocon: protein channel 5. Cleavage site where signal sequence is cut by a signal peptidase - As the secreted protein is starting to be made in the cytosol, we get the emergence of the signal sequence off the nascent (new) protein Cotranslational translocation SRP – signal recognition particle - Key: how does a secreted protein start? o You have a mRNA that is recognized by ribosomes, which will start making the protein? o As the protein is emerging off the ribosome, the first amino acids will contain a signal sequence o A signal recognition particle (SRP) will bind to it and move to the ER - SRP receptor and translocon are b
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