Protein SortingI.pptx

Protein Sorting I Molecular Biology Protein Sorting Proteins need to be localized in the proper place to perform their function Some proteins need to be localized to specific organelles while other need to be secreted outside cells. Protein localization is another way cells can regulate protein func...

Protein Sorting I Molecular Biology Protein Sorting Proteins need to be localized in the proper place to perform their function Some proteins need to be localized to specific organelles while other need to be secreted outside cells. Protein localization is another way cells can regulate protein function Protein Sorting Overview Protein Sorting Overview No signal  cytoplasm Localization occurs cotranslational ly ER Signal  ER localization Peroxisome Signal  Peroxisome Localization occurs after translation have been completed Localization in ER, Peroxisome and Mitochondria occurs previous to protein folding Mitochondrial Signal  Mitochondria NLS  Nuclear localization Nuclear Localization occurs after proper protein folding Protein Sorting Overview Secretory Pathway A pathway designed to : – Secrete proteins – Localize proteins to the plasma membrane – Add polysaccharides to proteins – Remove unfolded proteins – Send proteins to lysosome Secretory Pathway STEPS: 1. Protein synthesis and translocation across ER membrane 2. Protein folding and modification inside the ER lumen 3. Protein transport to the Golgi, Lysosome or exterior cell surface through budding and fusing of vesicles Endoplasmic Reticulum The rough endoplasmic reticulum is composed by ribosomes attached to membrane and translating proteins into the ER. Cotranslational localization Cotranslational localization refers to the fact that proteins entering the secretory pathway are translocated into the ER at the same time that they are being translated How do cells know which proteins should go to the secretory pathway? Because proteins that should go through the secretory pathway have a portion of their sequenced call the ‘ER signal’ This ER signal is composed of 6-12 hydrophobic residues, typically located in the N-terminus of the protein Cotranslational translocation (Signal Recognition Particle) Recognizes the ER signal sequence, stop translation and recruit the whole complex to the ER The interaction between SRP and ER signal sequence in achieved by hydrophobic interaction Cotranslational translocation (Signal Recognition Particle) Recognizes the ER signal sequence, stop translation and recruit the whole complex to the ER The interaction between SRP and ER signal sequence in achieved by hydrophobic interaction Cotranslational translocation (Signal Recognition Particle) Recruitment to the ER is achieved by the recognition of the SRP by the SRP receptor Both SRP and SRP receptor are weak GTPases but when bound to each other they undergo some conformation al changes that allows them to cleave GTP and obtain energy required for translocation Cotranslational translocation (Signal Recognition Particle) Cleavage of GTP releases enough energy to open the translocon and resume translation Cotranslational translocation (Signal Recognition Particle) As the nascent protein enters the ER lumen, the signal peptidase will recognize and remove the signal Cotranslational translocation (Signal Recognition Particle) Cotranslational translocation (Signal Recognition Particle) How about membrane proteins? Types of membrane proteins How membrane proteins get incorporated into membranes? In addition to signal peptide they also poses a ‘STOP TRANSFER’ anchor sequence, normally made of a helix made of hydrophobic residues, that disrupts translocation and localizes protein into the membrane How membrane proteins get incorporated into membranes? Often, in addition to the stop transfer anchor sequence they also have a stretch of positively charge residues that block translocation and tend to be positioned in the outside face of the ER How membrane proteins get incorporated into membranes? In the case of proteins whose N-terminus has to be located outside the ER membrane, the anchor sequence and the stretch of positively charged residues help position protein properly. In this case the stretch of positively charged residues are located before the ‘Stop Transfer’ anchor sequence. BIOINFORMATICS Based on our knowledge of signal peptides and anchor sequence we can use bioinformatics tools to predict whether or not a protein will be part of the membrane and the kind of disposition that will have once embedded in the membrane BIOINFORMATICS The use of hydropathy profiles can help us predict a membrane protein. Remember: both the signal peptide and the stop transfer anchor sequence are hydrophobic How do you get a membrane protein in the plasma membrane? Localize protein in ER membrane by co-translational translocation. Vesicle formation and fusion will bring it to the Golgi and from the Golgi it will be transported to the plasma membrane by vesicles How do you get a membrane protein in the plasma membrane? Localize protein in ER membrane by co-translational translocation. Vesicle formation and fusion will bring it to the Golgi and from the Golgi it will be transported to the plasma membrane by vesicles Problem You have found a protein domain that binds a virus. You reason that if you could put that protein domain in the surface of cells it would compete with the natural surface receptor of the virus and prevent its incorporation inside cells. Design a strategy to express this domain in the plasma membrane Why proteins in the secretory pathway pass through such intricate network of membrane organelles? (ERGolgiVesicle) Basically to undergo proper folding and maturation In the ER proteins undergo: – Glycosylation – Formation of disulfide bond – Proper folding – Assembly of multiple subunits – Proteolytic cleavage of sequences Glycosylation Very important for proper function specially for membrane proteins Usually involves addition of complicated branched polysaccharides Glycosylation Very important for proper function specially for membrane proteins Usually involved addition of complicated branched polysaccharides Glycosylation Very important for proper function specially for membrane proteins Usually involved addition of complicated branched polysaccharides Disulfide bond formation Usually only present in proteins to be secreted or to be place in the plasma membrane facing the exterior The reason is that the cytoplasm is reducing and the reaction requires and oxidant. Disulfide bonds are formed only in the ER where a series of oxidant factors catalyzes the reaction Folding and multimer formation Secretory pathway, overview