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Lecture 4: "Protein Synthesis & Transport"

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Biology 2382B
Robert Cumming

Cell Biology Lecture No. 4: Protein Synthesis & Transport st Monday January 21 , 2013 LECTURE 3 CONT’D Detecting Specific Proteins: -Western blotting (or immunoblotting) is a process used to detect specific proteins. First, an electric current is used to transfer the proteins on the SDS-polyacrylamide gel onto a membrane. This membrane is then incubated with primary antibodies that recognize your protein of interest. Enzyme- labelled secondary antibodies are then linked to the bound primary antibodies. Finally, a substrate is added to the membrane that is catalyzed by the enzyme and gives off luminescence (enhanced chemiluminescence), showing you where your protein of interest is located. This last step is very rarely used and more often fluorophores are attached to the secondary antibody. The luminescence makes an impression on the X-ray film and you can see darker banding patterns indicating higher expression. LECTURE 4 Protein Sorting & Protein Targeting: -In a typical mammalian cell, tens of thousands of different proteins are made and it becomes a major issue for the cell to direct them to the right destination. Targeting involves signal sequences directing proteins to their appropriate destinations (organelles) during or after synthesis. Sorting involves the directing of proteins involved in secretory pathways (that make it into the ER, Golgi body and lysosomes). General Principles & Overview Of Protein Synthesis, Sorting & Targeting: -Many proteins are synthesized by free-floating ribosomes in the cytosol that can be broken up into two categories: Proteins which function in the cytosol and proteins which are targeted (by a specific signal sequence) to function in intracellular organelles like the ER, mitochondria, chloroplasts or the nucleus. Other proteins are synthesized by ribosomes bound to the Rough Endoplasmic Reticulum and can also be broken up into two categories: Proteins which function in the ER and proteins which are sorted to function near the plasma membrane, Golgi body or lysosome. Accordingly, we see that two pathways arise for proteins: nonsecretory proteins and secretory proteins. -In the cytosol, mRNA transcript is recognized by the translational machinery and synthesized into protein. Once fully synthesized, some proteins are tagged with specific signal sequences which target the protein to mitochondria, chloroplasts, peroxisomes, or the nucleus. Other proteins have a signal sequence which targets the nascent polypeptide (as well as the associated ribosome) to the RER, where the signal sequence binds to a receptor and translation is completed in the lumen. Translocation channels (pores) help secretory proteins cross the pesky hydrophobic membrane (which takes energy to do). The Structure Of The Endoplasmic Reticulum: -The first part of the Endoplasmic Reticulum is actually a direct continuation of the nuclear membrane, though they do become a collection of stacked tubules (cisterna) that are distinct from the nuclear membrane. The association of ribosomes with its membrane is what defines the RER. Studies On The Secret Lives Of Secretory Proteins: -A radio-labelling experiment was performed to answer how certain proteins are secreted. This included using amino acids with a radioactive signature that are taken up by a cell culture and incorporated in the building of proteins. After homogenization, it was discovered that open membranes of RER fragments have a tendency to re-form and become microsomes. Sucrose density-gradient centrifugation was then used to concentrate the microsomes (RER matter). Half of this centrifuge was treated with detergent (to break apart microsomal membrane) and protease (to break apart proteins inside) and half was treated with just protease. The conclusion of this experiment is that secretory proteins need to enter the RER, where proteins are either made or imported. Functions Of The Rough Endoplasmic Reticulum: -Secreted and membrane-bound proteins are sorted through the RER. Post-translational modification such as the adding of sugars and carbohydrates to polypeptides is performed in the RER. Other structural features such as disulphide bonds are added in the RER as well. Chaperones assist newly translated polypeptides to fold correctly in the RER. The Simultaneous Occurrence Of Translation & Translocation: -Another experiment was performed to determine if proteins are made, then imported into the RER or if they are made and imported simultaneously. In a cell-free system (in vitro protein translation system), the mRNA of a known secretory protein is translated. When these completed proteins with their signal sequences were introduced to microsome membranes, they wouldn’t enter. The cell-free experiment demonstrated that translocation of secretory proteins into microsomes is coupled to translation. Major Players In The Rough Endoplasmic Reticulum: -The major players associated with the RER include: the amino termin
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