2. Entering the ER: Translocation of proteins across the membrane of the ER is usually studied using microsomes (vesicle

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2 Entering The Er Translocation Of Proteins Across The Membrane Of The Er Is Usually Studied Using Microsomes Vesicle 1 (94.7 KiB) Viewed 23 times

2. Entering the ER: Translocation of proteins across the membrane of the ER is usually studied using microsomes (vesicles made from rough endoplasmic reticulum). Microsomes of the rough ER carry ribosomes attached to their outer surface. Translocation of proteins across the microsomal membrane can be assessed by several experimental criteria: (1) the newly synthesized protein is protected from added proteases, but not when detergents are present to solubilize the protecting lipid bilayer; (2) the newly synthesized proteins are glycosylated by oligosaccharide transferases, which are located exclusively in the lumen of the ER; (3) the signal peptides are cleaved by signal peptidase, which is also active only on the luminal side of the ER membrane. You want to use these criteria to decide whether the protein synthesized from a purified mRNA is translocated across the microsomal membranes. Therefore, you translate the mRNA into protein in a cell-free system in the absence or presence of microsomes. You then prepare samples from these translation reactions in four ways: 1.) no treatment, (2) add a protease, (3) add a protease plus a detergent, (4) disrupt microsomes and add endo-glycosidase H (endoH), which removes N-linked sugars that are added in the ER. See gel below for results: Microsomes absent Treatment Protease Detergent + protease endoH Protein Migration: Large Small Microsomes present 1 2 3 4 5 6 7 8 a. Explain the experimental results that are seen in the absence of microsomes (lanes 1-4). Grading rubric: a. Why are proteins present in lanes 1 and 4 but not 2 and 3 (1 point)? b. Explain the migration of the proteins in lanes 5,6, and 8. Rubric: Protein migration in lanes 5,6, and 8 based on your knowledge of protein translocation and processing in the ER (1 point). c. Using the three criteria outlined in the problem, decide whether the experimental results in the presence of microsomes (lanes 5-8) indicate that the protein is translocated across microsomal membranes. Is the protein anchored to the membrane, or is it translocated all the way through the membrane? Rubric: Use your analysis from "b" to decide whether the protein is found in the lumen or membrane of the ER (.5 points).