D103 Lecture 11.2- PTMs - Bio D103 Cell Biology Instructor...

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Bio D103: Cell Biology Instructor: Dr. Scott Atwood Fall 2016
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How does the absence of a preclass quiz influence your reading of the textbook? A. More likely to read the textbook B. Less likely to read the textbook C. No change in reading habit since I need the reading to understand the lectures iClicker question 11-0
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APEX tagging facilitates proteome research APEX (engineered ascorbate peroxidase): Fused to a protein of interest Introduced into a cell Exposure to biotin-phenol and hydrogen peroxide causes APEX to covalently tag surrounding proteins with biotin- phenoxyl radical oxidation product Cells are lysed, biotinylated proteins are purified with streptavidin beads, and identified by mass spectrometry
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Mitochondrial staining of APEX-tagged proteins IMS-APEX found in inter- membrane space of mitochondria IMS-APEX found in matrix of mitochondria western blot using streptavidin antibody Ponceau staining is similar to Coomassie, stains all proteins H2O2 = hydrogen peroxide, BP = biotin- phenol
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Comparison of mass spectrometry results with known mitochondrial proteins sample mass spectrometry analysis shows distribution of mitochondrial proteins in human genome (1st column) and APEX interaction proteins with IMS annotation (2nd column)
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Immunofluorescence with novel mitochondrial proteins Immunofluorescence of novel mitochondrial proteins from APEX-tagging that are fused to the V5-tag mito-GFP marks mitochondria
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Immunofluorescence with novel mitochondrial proteins Discussion : Does a hit in an APEX tagging experiment mean the two proteins are interacting?
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After reading the paired chapters , you should be able to: Understand the concept of post-translational modification Compare and contrast phosphorylation versus ubiqutination After reviewing this lecture , you should be able to: Understand the roles of PTMs Describe how phosphorylation occurs and its functions Describe how ubiquitination occurs and its functions Lecture 11: Learning objectives
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Protein complexity presents a unique problem in cell biology
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reversible
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Post-translational modifications (PTMs) of p53 and histones
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