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Download Major Protein-sorting pathways in eukaryotic cells
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Major Protein-sorting pathways in eukaryotic cells Key elements for the Protein targeting events 1. Signal sequence: the amino acid sequence of a protein that provides the information to target the protein to a particular organelle. 2. Receptors for the signal sequences. 3. Translocation channels that allow transfer of proteins across the membrane bilayer. 4. Energy that drives unidirectional transfer across the membrane. Where are the endoplasmic reticulum, Golgi, and mitochondria? Synthesis of secretory proteins and their cotranslational translocation across the ER membrane Wenxia: Wenxia: 1.1. ER ERsignal signalsequence sequence emerges emerges 2.2. The Thebinding bindingby byaa signal-recongition signal-recongition particles particles(SRP) (SRP) 3.3. SRP SRPdelivers deliversthe the ribosome/nascent ribosome/nascent polypeptide polypeptide complex complextotothe theSRP SRP receptor receptorininthe theER ER membrane, membrane,and and GTP binding GTP binding Signalrecognition particles Wenxia: Wenxia: 1.1. Six Sixsubnits subnits 2.2. All Allproteins proteinsbinding bindingtoto 300-nucleotide 300-nucleotideRNA RNA except p54 except p54 3.3. P54 P54bind bindtotoER ERsignal signal sequences and have sequences and have hydrophobic hydrophobicbinding binding groove groove Translocon Wenxia: Wenxia: 1.1. First Firstfound foundininyeast yeastby by mutations mutations 2.2. Sec61 Sec61complex, complex, consisting consistingofof33proteins proteins 3.3. Sec61a Sec61acontacts contacts translocating translocating polypeptide polypeptidechain chain 4.4. AAcylinder cylinder5-6 5-6nm nmhigh, high, 8.5 nm in diameter, 8.5 nm in diameter,and and pore 2 nm diameter pore 2 nm diameter 5.5. Open Openand andclose closeofof translocon transloconisisregulated regulated Major topological classes of integral membrane proteins synthesized on the rough ER Wenxia: Wenxia: Hydrophobic Hydrophobic transmembrane transmembrane segment segmentisisthe the stop-transfer stop-transfer anchor anchorsequence sequence Synthesis and insertion into the ER membrane of type I single pass proteins Type II singlepass proteins Experiments: Experiments: Mutating Mutatingthe the positive charged positive charged amino aminoacids acids change changethe the orientation orientationofofthe the protein. protein. Arrangement of topogenic sequences of different membrane proteins GPI anchored proteins Mitochondrial Protein import into the mitochondrial matrix Tom-translocon of outer membrane Tim-translocon of the inner membrane Wenxia: Wenxia: Targeting Targetingsignals signals Signal Signalreceptor receptor Translocation Translocation channel channel Energy Energy Transporting protein from the cytosol to the inner mitrochondrial membrane Transporting proteins from the cytosol to the mitochondrial intermembrane space How to analyze protein movement into membranes and organelles The in vitro system How to identify signal sequences Genetic manipulations 1. Sequence homology 2. Targeted mutagenesis 3. Fusion proteins Hydropathy profiles How to identify a translocon component that contacts nascent secretory proteins Chemical cross-linking