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Protein Sorting and Membrane Trafficking for zoology 570

Protein Sorting and Membrane Trafficking for zoology 570 -...

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Protein Sorting and Membrane Trafficking -macromolecules are NOT randomly distributed throughout the cell *different organelles have different proteins (and other macromolecules) *with cytoplasm, macromolecules are also nonrandomly distributed *even within a given structure/organelle different macromolecules are often differently distributed 3 General Mechanisms used for sorting proteins 1. Sorting before translation -information is found in mRNA, mRNA is targeted to particular place in cell 2. Sorting DURING translation -sorting information is in nascent protein (i.e. protein in process of being translated) -when information “appears” during translation, the mRNA-ribosome-nascent protein Complex is target to particular place in cell 3. Sorting after translation -sorting information is contained within completely translated protein -AFTER translation, this information is used to target protein to destination 1. Sorting Before Translation (cytoplasmic) -idea is simple- cell puts mRNA in a particular place in cytoplasm, mRNA is translated there, thus the protein accumulates in that place -originally thought to be an uncommon way to sort proteins but is now looking quite common, in analysis of 3370 different mRNAs, approximately 70% were found to be targeted to specific regions of cytoplasm -Best known examples include mRNAs encoding proteins involved in: *development, cell motility, cell polarization Sorting of Beta-actin before translation *most nonmuscle cells have two actin isoforms gamma-actin and beta-actin *each is product of different gene, proteins are VERY similar- differ only in four amino acids out of 375 -Distribution of Gamma and Beta-actin in Fibroblasts **migrating fibroblasts *Gamma-actin is distributed throughout cell Beta-actin is distributed at the leading edge of the cell (i.e. the edge that faces the direction of movement)
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Question: How is Beta-actin localized to leading edge? Answer: -Beta-actin mRNA is localized to leading edge -Beta-actin mRNA has RNA “zipcode” in its 3’UTR *zipcode is 54 NT sequence that binds to protein called ZBP1 **ZBP1= zipcode binding protein 1: anchored to plasma membrane at leading edge by unknown mechanisms. Anchoring of B-actin mRNA at leading edge means that B-actin mRNA is translated at leading edge so protein accumulates there EXPERIMENTS: 1. Purifying B-actin Proteins -label B-actin with fluorophore and inject it into fibroblasts -compare distribution of injected B-actin to endogenous B-actin (B-actin already there) Result: injected B-actin does NOT localize to leading edge even though endogenous B-actin does. This implies that information that gets endogenous B-actin protein to leading edge is NOT contained within B-actin protein itself 2. Look at Distribution of B-actin mRNA and Gamma-actin mRNA Result: B-actin at leading edge Gamma-actin distributed throughout cytoplasm 3. Try to deduce basis of mRNA targeting using fusion RNAs B-actin mRNa B-galactosidase mRNA: allows you to specifically see the fusion
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Protein Sorting and Membrane Trafficking for zoology 570 -...

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