Question

Please answer letter B!

1. (1.5 pts) 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 micrasomes. 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 Microsomes Treatment Protease Detergent present --+- + protease endoH Protein Migration: Large Small 5 6 7 8 a. Explain the experimental results that are seen in the absence of microsomes (lanes 1-4 Grading ubric a why are protein present in lanes 2 and 4 but not 2and 3 105 pont? b. Explain the migration of the proteins in lanes 5,6, and 8. Nubric Protain migration in lanes54 and baed on your knowledige of probai tramlocation and procnsing in the ER (05 paint Problem set 8: Insertion into the ER and Vesicular Trafficking 1(1.5 pts) Entering the ER: Translocation of proteins across the membrane of the ER is usually studied uning microsomes (vesicles made from rough endoplasmic reticulum). Microsomes of the rough ER carry ribosomes attached to their outer surface. Translocation of proteins across the microsomall 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 solubilire 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 dleaved 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 cel-free system in the absence or presence of microsomes. You then prepare samples from these translaton reactions, four way 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. Seegel below for results Microsomes absent Treatment Protease Detergent»-- +protease endoH present Protein Migration Large Small 5 6 7 8 a. Explain the experimental results that are seen in the absence of microsomes (lanes 1-41·M㎎-a. why utmws present in lanes1 and 4 but nat 2 and5point? b. Explain the migration of the proteins in lanes 5,6, and 8. Mit noun-guion n'anes Sitbewd-w๒blpdptm and prooessing in the ER ID

Answer 1

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