BIOMG 1350 – Section 5 Homework (Due at the beginning of your section) 1 Name: Protein Sorting at the Endoplasmic Reticulum Read the following sections in ECB Chapter 15: Proteins enter the ER while being synthesized Soluble proteins are released into the ER Lumen Stop and start signals determine the arrangement of a transmembrane protein in the lipid bilayer Also read 'GEL ELECTROPHORESIS' (ECB3 Panel 4-6 or ECB4 Panel 4-5). Introduction: Peter Walter (one of the authors of your textbook) was interested in understanding the mechanism that allows some proteins to be synthesized on cytosolic ribosomes, while other proteins are synthesized on membranes of the endoplasmic reticulum (ER) where they enter the secretory pathway. He knew that proteins destined for the secretory pathway have a 'signal sequence' which is necessary for them to be targeted to the endoplasmic reticulum. But how is the sorting achieved? Peter approached this question biochemically and discovered SRP (the Signal Recognition Particle). In this section, we are going to go over simplified versions of his experiments to see how he discovered the properties of the particle. Peter used a technique called in vitro translation followed by gel electrophoresis to study the synthesis of proteins. Since the cytosol contains all of the components required for protein synthesis, it can be extracted from cells and used to synthesize proteins in a test tube…no cell needed (this is in vitro translation)! The cytosolic extract in these experiments is lacking mRNAs and membranes, so one can synthesize a particular protein of interest simply by adding the corresponding mRNA to the cytosolic extract. The mRNA will be recognized and synthesized by the ribosomes and other cytosolic components. In order to analyze the product of this in vitro translation experiment, one can perform polyacrylamide gel electrophoresis (PAGE) on the finished reaction. PAGE allows you to determine the size of any proteins in the finished reaction.BIOMG 1350 – Section 5 Homework (Due at the beginning of your section) 2 Experiment #1: In step 1, Peter added two mRNAs to the extract (sample 1). One mRNA encoded the cytosolic protein hemoglobin, and the other the secretory protein pre-prolactin (a hormone that is normally made on the endoplasmic reticulum and contains a signal sequence). He of course also had a control sample in which no mRNA was added (sample 2). Finally, He subjected the finished reaction to gel electrophoresis (step 2) and analyzed the results (step 3). 1. Can pre-prolactin be synthesized in the absence of membranes? Experiment #2: Now that Dr. Walter had found that a protein normally secreted can in fact be synthesized in the absence of endoplasmic reticulum, he wondered what would happen if he added SRP and endoplasmic reticulum (ER) membrane vesicles to the reaction before adding mRNA. The ER membrane vesicles he used are derived from the endoplasmic reticulum of cells and are sealed so that the only way a protein can get into them is being translocated across the membrane.BIOMG 1350 – Section 5 Homework (Due at the beginning of your section) 3 He ran two reactions one without membrane vesicles (as above) and one with SRP and ER membrane vesicles. To both he added the two mRNAs. He analyzed the results by gel electrophoresis. He obtained the result shown on the right. He noticed that the migration of the pre-prolactin protein was faster in the presence of SRP and ER membranes than without them. 2. What does this tell us about the size of the protein product in sample 2 with ER membranes? 3. Why is the size of pre-prolactin changed, but the size of hemoglobin is not (HINT: read below for more info)? Pre-prolactin has a signal sequence at its N-terminal end. This directs it to the endoplasmic reticulum. Peter could show that the smaller protein product in the presence of SRP and ER membrane vesicles was in fact the mature protein with the signal sequence cleaved off! In addition, the protein was found in the lumen of the ER membrane vesicles! He had reconstituted translation, translocation into the ER, and removal of the signal sequence in a test-tube reaction as diagrammed below! Since the signal sequence is removed, the protein in now called prolactin. In the section, we will discuss further experiments that Peter did that revealed how SRP works. Be sure you understand the experiments described above as the section activity builds on