D103 - Lecture 11 - Protein Sorting II.docx

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Biological Sciences
Grant Mac Gregor

A. Writing questions a. What if we mutate this protein? B. New knowledge nugget a. Gel electrophoresis B. Clicker a. Mutation in nDNA leads to a complete block in mito protein import i. Which protein could be affected by this mutation 1. Porin a. Not related to protein import 2. Electron transport complex 3. Mitochondrial translocation machinery in the inner membrane 4. Enzyme that cleaves the mitochondrial sorting signal a. Cleavage process can still occur without sorting signal removal 1. None of these conditions above prevent mito protein import completel a. How to completely block protein import? i. Mutate TOM complex 1. Always needed i. Mutate chaperones - proteins get folded B. Intermediate Filaments a. Sequence conservation i. Have similar amino acids (same properties) ii. Low sequence conservation 1. Between 70 intermediate filaments 2. More similar between IF classes b. Conserved domains i. N-terminal domain ii. Central rod-like domain iii. C-terminal domain B. Reconstitution Assay a. Purified protein with signaling signal b. WT mitochondria c. Mix and observe protein import i. Add protease to mixture 1. Protease will not be able to digest imported proteins b. Mechanistic insight i. Change components of the assay b. Reconstitute a process in a test tube c. Requirements i. N-terminal sorting signal ii. Energized mitochondria iii. Unfolded protein iv. Sorting signal is removed 1. Initial protein is larger than imported protein B. Sorting signals - targets proteins a. Mitochondrial matrix i. Amphiphatic helix 1. Removed after arriving in the matrix 2. Sorting signal ii. Outer/Inner Membrane 1. Internal sequences 2. Not removed b. All mitochondrial sorting signals i. Necessary and sufficient for mitochondrial import ii. Recognized by specific mito receptors b. Mitochondrial proteins are mostly imported B. Hsp70 a. Heat shock protein b. Active during heat c. Use ATP to keep it in an unfolded state B. Mitochondrial Protein Import a. TOM i. Translocation outer membrane channel b. TIM i. Translocation inner membrane channel b. Import receptor is located next to the import channel c. TOM and TIdownM are found where the outer and inner membrane are close together d. Has a pulling protein that provides directionality to the process i. Using ATP, to pull into matrix ii. Similar to Ran in nuclear import b. IGNORE SUBUNITS OF THE TIM/TOM c. Protease that cleaves off the imported sorting signal d. Protein needs additional chaperones to be folded correctly
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