BICD 110 Wi12 Problem Set#2

BICD 110 Wi12 Problem Set#2 - 1) Translocation of proteins...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: 1) Translocation of proteins across rough microsome membranes can be judged by several criteria: 1) Translocated proteins are protected from proteases, unless the membranes are solublized by detergent treatment. 2) Newly synthesized proteins are glycosylated by oligosaccharide present in the ER lumen. 3) The signal peptide is cleaved by signal peptidase which is only on the luminal side of the ER. Use these criteria to determine if a protein is translocated across microsomal membranes. mRNA is in vitro translated in the presence of microsomal membranes and then samples are treated in one of four ways: 1) No Treatment 2) Protease 3) Protease + Detergent 4) microsome disruption + endoglycosylase H (endoH) which removes N linked sugars These samples are run on an SDS-PAGE gel and the following results are obtained: 1 2 3 4 5 6 7 8 A) Explain the experimental results that are seen in the absence of microsomes (lanes 1-4). B) How do you account for the migration patterns seen in lanes 5-8. C) Is the protein anchored in the membrane of is it translocated all the way through? Explain your reasoning. 2) A transmembrane domain protein is fused to Alkaline Phosphatase (AP) and translated in the presence of microsomes. Following translation, the activity of AP is measured and the following results were obtained (A is an internal hydrophobic sequence): A) Draw the orientation of each of these proteins in the microsome, clearly label the cytosolic side in each case. B) For hydrophobic domain A indicate where positively charged amino acids would be located. C) In order to confirm your topology, you use site directed mutagenesis to generate three different constructs each with a novel N-linked glycosylation site that you created: Draw the expected SDS-PAGE results for each of the three constructs showing the size of the protein with and without EndoH treatment. 3) A common method for blocking energy dependent steps is to introduce a nonhydrolyzable nucleotide analog into a cell or biochemical reaction (ATP-gammaS, GTP-gammaS, etc). Describe what would happen if the following nucleotide analogs were introduced into the following processes. Include a diagram showing where the process arrests. Process Nucleotide A) LDL Receptor Endocytosis ATP-gammaS B) LDL Receptor Endocytosis GTP-gammaS C) Protein Translocation into the ER GTP-gammaS (assume that this does NOT interfere with the ribosome or translation) D) ER-Golgi Trafficking (anterograde) ATP-gammaS E) ER-Golgi Trafficking (anterograde) GTP-gammaS...
View Full Document

This note was uploaded on 02/01/2012 for the course BIOLOGY 110 taught by Professor Jameswillhelm during the Spring '11 term at UCSD.

Page1 / 13

BICD 110 Wi12 Problem Set#2 - 1) Translocation of proteins...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online