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Lecture 4

Lecture 4 - Protein sorting.docx

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Biology 2382B
Robert Cumming

Lecture 4 - How proteins know where to go Protein sorting/targeting  Typical mammalian cell: o 10,000+ different proteins o Must go to the right destination – must be localized correctly  Newly made peptides must be directed to the correct destination  Targeting: o Directing proteins to the right destinations (organelles) o During or after synthesis  Sorting: o Direct proteins to the secretory pathway (ER, Golgi, Lysosomes) o More related to proteins involved in the secretory pathway – proteins which make it inside the ER, Golgi and lysosomes (Secretory, vesicles, getting rid of stuff) 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)  Most of these targeting sequence happen at the amino terminus o Other proteins are synthesized by ribosomes attached to ER (the rought ER)  Those which reside in ER and proteins which are sorted to Pm, Golgi complex and lysosomes o Accordingly, Two major protein sorting pathways are known: nonsecretory and secretory  Many proteins are made in the cytosol o Some proteins which have unique sequence of amino acids which can be recognized by other proteins to take them to the right organelle  Makes the protein first and then you read the signal to direct it o Some poly peptide chains have signal sequences as well but are required to take them to the ER where translation of the protein continues to occur as the polypeptide is still being formed  The newly emerging poly peptide chain is directed to the ER and from there you continue to translate and the new polypeptide chain, in continuing to be formed, is placed directly in the lumen of the ER ER Structure  The ER extends directly from the nuclear membrane o Area closest to the Nucleus is actually connect to the nucleus  Cisterna – tubules which tend to be stacked on top on top of each other Lecture 4 - How proteins know where to go  As we move away from the nucleus there’s space between the tubules – the many dots between the different tubules are ribosomes which is what makes it the “rough” ER  Stringing the concepts together! – how did we figure this structure out? o Identify cellular features by microscopy, isolate and homogenize them to free organelles o Sucrose density gradient centrifugation of homogenate allows for isolation of microsomes and ribosomes o SDS page is used to identify the newly formed proteins  How do secretory proteins enter the ER?  How are things secreted? o Tested for by adding radio labeled amino acids which are then taken up by some amino acids o You then disrupt the cell o Microsomes formed – the smaller bits of the ER which have broken apart and reformed on itself – you now have miniature version of ribosomes and still have ribosomes associated around the microsomes o You take that mixture of microsomes and ribosomes and half treat with detergent and then and protease  The detergent disrupts the membrane and then add the protease which chews apart the stuff inside the cell o With the other half you only add protease  This is where only the ribosomes will be eaten on the outside nothing on the inside  Demonstrated that you have to get inside the ER for a protein to be secreted – secretory proteins must go through the ER ER functions  Secreted and membrane proteins are sorted through the Rough ER  Sugars/carbohydrates are added to the polypeptide  Disulfide bonds are formed  Proteins are folded by “chaperones Translocation and translation occur simultaneously  Next question – we know that the secretory proteins must get into the ER, so are they made first and then in the ER are made I the ER?  In vitro protein translation system – being made in a test tube o Pure messenger RNA which is then added to a concoction to form completed proteins with signal sequences for protein which is known to be secreted o Add microsomes o If you have completed proteins with signal sequences nothing would be incorporated Lecture 4 - How proteins know where to go  If you mixed everything together – in vitro translation system along with mRNA with Microsomes as well, than you get incorporation of mature protein chain with out signal sequence  This proved that translation and translocation is happening at the same time RER: What are the major players?  Amino terminal signal sequence of newly initiated polypeptide (nascent proteins  Signal recognition particle (SRP)  SRP receptor embedded in ER membrane  Translocon: protein channel  Cleavage site which cuts off signal sequence Co-translational translocation  first few amino acids at the amino terminus will have a signal sequence for proteins which are destined for secretion  This signal sequence is then recognized by the SRP which then directs it to towards the SRP receptor (Directs the ribosome + polypeptide complex)  The binding of the SRP complex and the SRP receptor found at the ER membrane is enhanced when it is bound to GTP o GTP therefore binds to the SRP to help with the binding of SRP to SRP receptor  Once the GTP SRP binding has happened and the SRP binds to the SRP receptor, the SRP receptor is also close to the translocon (It’s a pore! Like a large donut, at the center of the donut where
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