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BIOL 201 - Lectures 16 to 18

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Biology (Sci)
BIOL 201
Greg Brown

Lecture 16 There are three different types of targeting systems to determine the organization of a protein in a membrane. Multi-pass proteins: Combinations of 3 types. Hydropathy profiles • Characteristic of a given protein • Graphic way of identifying hydrophobic regions in the protein that can serve as transmembrane domains, signal sequences, signal anchor sequencesor stop-transfer sequences. ⇨ Determine average hydropathy index for 5 amino acidwindow ⇨ Move window over one amino acid, determine again ⇨ Plot hydropathy average vs. position of amino acidin polypeptide Positive amino acid sequences flanking signal sequences help predict protein orientation ⇨ Understanding the characteristics of the polypeptide chain allow you to determine the orientation of a particular membrane protein (n-terminus in lumen vs in cytosol). Post-translational modifications in the ER As proteins are being synthesized and enter the ER,they undergo a large number of processes. (Newly made proteins in the ER membrane and lumen undergo modification) ⇨ Disulfide bond formation ⇨ Folding ⇨ Glycosylation ⇨ Assembly into multimeric proteins ⇨ Only properly folded and assembled proteins can exit the ER and pass through Golgi to final destination. “quality control” Correct disulfide bond formation • Form between cysteine residues. The oxidation of sulfhydryl groups in successive cysteine residues in a polypeptide chain results in the formation of disulfide bridges. • PDI mediates this process. • (PDI = Protein disulfide isomerase) • Oxidized PDI Protein with cysteines comes into the ER. Oxydized PDI will react with successive cysteines creating a disulfide bridge. • ERO1 will react with reduced PDI and convert it back to its oxidized form, reducing the sulfhydrides which oxidize spontaneously in the ER with molecular oxygen. Some disulfide bonds may need to be rearranged. Incorrectly formed disulfide bonds are exposed and can react with reduced PDI which will mediate the breaking and reforming o disulfide bonds to stabilize the proper form of the mature polypeptide. Protein glycosylation in the ER • Most plasma membrane and secreted proteins containattached carbohydrate chains • Glycosylation may be N-linked or O-linked • N-linked: A preformed oligosaccharide is added to Asn (Asparigine)residues of many proteins in the ER, as they are synthesized - Added only at Asn residues flanked by X-Ser or X-Thr (C-side) • O-linked: Attachment of carbohydrate residues (often complex) to syrine or threonine. • Oligosaccharide: (Glc) 3Man) (9lcNAc) for2ed on Dolichol phosphate, an ER phospholipid.First step in formation of Dolichol oligosaccharide inhibited by tunicamycin • Tunicamycin is an antibiotic, a very useful tool to observe the fate of the protein if it is not successfully modified.) • Oligosaccharide processed prior to passage to Golgi: quality control Quality control: oligosaccharide modification & chaperone proteins (Calnexin, Calreticulin) Capable of binding to oligosaccharide with final glucose. Unfolded protein response • BiP = Hsc 70 protein of ER lumen, facilitates protein folding. • Unfolded proteins bind to BiP, release BiP from Ire • Unbound Ire dimerizes ▯ ac▯vates endonuclease • Endonuclease cleaves Hac1 mRNA, allows production of Hac1 protein Usually occurs during heat shock • Hac1 enters nucleus, stimulates transcription of folding proteins Mitochondrial protein import mainly post-translational • You can take your protein and synthesize it in your cell-free medium. Mitochondria not necessary for protein synthesis. However, mitochondria needed a functional transmembrane potential to take up proteins. Mitochondrial signal sequences • Most proteins that go to the mitochondria have a sequence that targets them to the matrix. - Have N-terminal signal sequence that is removed bypeptidase • Outer membrane, some inner membrane: internal signal sequences • Signal sequences necessary and sufficient for targeting. Attaching a signal sequence to any protein will direct it to the mitochondrion. • Matrix signal sequences: amphipathic helix Hydrophobic and Hydrophilic regions. Positive amino acids are on one side of the helix, and hydrophobic amino acids are on the other. Hydrophobic AAs attach to a hydrophobic crevice in a receptor on the outer mitochondrial membrane. Protein import into matrix: chaperones and receptors • Newly made protein associates with chaperone (Hsc70). Energy from ATP hydrolysis keeps protein unfolded. • Targeting sequence associates with import receptor. • Precursor transferred to Tom 40 import channel in outer membrane, associates with Tim channel in inner membrane • Protein drawn into matrix by proton motive force, associates with matrix Hsc70 • Targeting sequence cleaved, association with Hsc60 (some) Targeting to the inner membrane - Path A and path B both feature matrix-targeting signal sequences. Interact with a receptor on the outer membrane. Go from TOM 40 translocon to TIM23-TIM17 translocon. Protein either has an internal stop- transfer sequence which stops protein transfer at TIM23-17 and the protein is released. C terminus in intermembrane space, n-terminus in the matrix. Second pathway used by proteins that are often membrane proteins, components or respiratory pathways. Enters matrix completely and associates with Oxa1. Allows for proper assembly and orientation. A third pathway is used by ATP/ADP translocase andother proteins with multiple transmembrane domains. They have internal targeting sequences, but never reach TIM23-17, but rather reach another translocon which allows them to orient properly. Inter-membrane space targeting • Precursors have “bi- functional” presequence: matrix targeting + IMS targeting • Precursor enters like matrix protein. Matrix targeting seq cleaved • IMS targeting seq stops in I.M. during import, dissociates from Tim channel, IMS targeting sequence cleaved • Outer membrane: matrix targeting but “stop transfer” sequence anchors in outer membrane before transfer to matrix Lecture 17 Overview of major protein transport routes in the secretory pathway From ER to Golgi: A protein in the ER can be transmitted to a Cis-Golgi network, and then to the trans-Golgi network through Golgi apparatus. This is mediated by cisternal progression. From Golgi to ER: Once it reaches the trans-Golgi network is either sorted to plasma membrane, or to the lysosome. What allows soluble and membrane proteins to be moved accurately and efficiently within the secretory pathway? Vesicles carry proteins through the system. Three well-characterized types of transport vesicles that work in different transport steps - Clathrin - COP2: From ER to Golgi - COP1: From Golgi to the ER Vesicles of the same type have a relatively uniqueshape and size Unique morphology. Each type of vesicles is associated with differentcoat proteins covering their sytosolicsurface Very small GTP binding proteins also located on coat protein surface. The role of coat proteins 1. Vesicle formation: shape and assemble a vesicle into high curvature of the membrane that is typical of a transport vesicle in ≈70nm in diameter 2. Cargo selection: determine which proteins are incorporated into shaped vesicles Formation of vesicles is initiated by a family of coat-forming small Arf GTP binding protein 1. Sar1 for COPII vesicle formation in the ER membrane 2. Arf1 for COPI vesicle formation in Golgi 3. Arf6 for Clathrin vesicle formation in trans-Golgi network or plasma membrane Arf GTPasec ycles between GDP- bound (inactive) and GTP-bound (active) forms How the cell can initiate the formation of a COPIIvesicle. 1. Sec12, a specific Sar1 GEF embedded in the ER, recruits cytosolic, inactive Sar1-GDP, and switches it into active Sar1- GTP; once activated, the N-terminal hydrophobic helix of Sar1- GTP is exposed and inserted into the ER 2. Membrane-bound Sar1-GTP recruits Sec23-Sec24. Sec24 interacts with membrane cargoes and cargo receptorsso cargo proteins are recruited into forming vesicles 3.
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