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UT BIOL 3030 - Trafficking & the Endomembrane System I

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Lecture 13: Trafficking & the Endomembrane System I- Major Protein-sorting Pathways in Eukaryoteso Signal-based Targeting vs. Vesicle-based traffickingo Targeting: occurs during or soon after translation, beginning on ribosomes in cytosolo Secretory pathway: proteins are moved from ER to their final destination via membrane-enclosed vesicleo Moving proteins across ER can be done directly from ribosomes moving them into ER where they are folded and post-translationally modifiedo Targeting sequences  signal sequences/peptides that signal movement- All eukaryotic cells have ERo ER consists of convoluted tubules and flattened sacs continuous with membrane of the nucleus Smooth ER is location of lipid synthesis Rough ER is location for first step in secretory pathway with attached ribosomes Synthesis location of membrane and soluble proteins Protein synthesis in the ER- ER membrane-bound and free ribosomes are identical  membrane bound ribosomes are recruited during synthesis of a polypeptide containing an ER signal sequence- Secretory Proteins enter the ER lumeno Pulse-chase experiments: demonstrated that secretory proteins are localized in ER lumen after synthesis- Translation and Translocation occur Simultaneouslyo An ER targeting sequence in the nascent protein directs the ribosome to the ER and initiates translocation of the growing polypeptide across the ER membrane ER targeting sequence is located at hydrophobic N-terminus of protein Its first part to emerge from ribosome Cleaved after directing ribosome docking on the ERo Translocation of secretory proteins in ER lumen is coupled with translation- Signal Recognition Particle (SRP)o SRP binds to hydrophobic core of ER signal sequence and large ribosomal subunito Recruits ribosome to SRP receptor system, docks of ER translocon and cotranslationally inserts nascent protein into/through ER membrane- Sec61a is a translocon componento Complex that contacts nascent secretory proteins as they pass through translocon into ER lumeno Translocation is driven by translation elongationo Translocating peptide is needed to open Sec61, otherwise it is remains impermeable (-) peptide  channel closed by plug that moves out during protein translocation (+) peptide  ring of residues at constricted waist  forms gasket that keeps channel sealed to small molecules- Post-translational translocationo Some proteins enter ER after translation so no SRP/receptor involvement occurso N-terminus enters ER  signal peptidase cleaves signal sequenceo BiP and Sec63 complex enforce unidirectional translocationo BiP ATP hydrolysis powers protein translocation - Insertion of Membrane Proteins into ERo Membrane protein topology: number of times a polypeptide spans a membrane and its orientationo Topogenic sequences: direct the insertion of nascent proteins into ER membrane N-terminal signal sequences Stop-transfer anchor sequences Signal-anchor sequences- Membrane insertion/orientation of Type 1 single-pass transmembrane proteinso Topology: N-terminus in ER lumen, single transmembrane, C-terminus in cytosolo Elongation continues until stop-transfer anchor sequence enters translocon- Membrane insertion/orientation of Type 2/3 single-pass transmembrane proteinso Topology: Lack N-terminal SS, have single internal hydrophobic signal-anchor sequence  ER signal sequence AND membrane anchoro Orientation of charged residues dictates orientation of signal anchor sequence- Membrane insertion of tail-anchored proteinso C-terminal tail-anchored proteins No N-terminal signal sequence Hydrophobic C-terminus  not available for insertion until protein synthesis is completeo Get3-ATP receive nascent protein and docks onto ER membraneo Get3-ATP hydrolysis and ADP release  releases nascent protein into Ge1/Get2o Get1/Get 2 receptor releases tail-anchor sequence into ER membrane- Topogenic sequences determine orientation of ER membrane proteinso Internal stop-transfer anchor sequence  N-termini = exoplasmic sideo Internal signal-anchor sequence  N-termini = cytoplasmic side- Hydropathy profileso Predicts likely topogenic sequences in integral membrane proteins Y-axis (+) values  relatively hydrophobic region  likely transmembrane domain Y-axis (-) values  relatively hydrophilic region- GPI-anchored proteinso Amphipathic GPI molecule  anchor covalently attached protein in membrane GPI anchored protein formation- Protein is synthesized and inserted into ER like type I- Specific transamidase  cleaves protein within exoplasmic-facing domain, near stop-transfer anchor sequence - Covalently links new C-terminus to terminal amino group No cytoplasmic domain  can more around b/c not anchored- Protein Modifications, Folding, and Quality Control in ERo Oligosaccharides (allows for cell-surface interactions and communications) O-linked oligosaccharides  -OH on serine/threonine N-linked oligosaccharides  -NH2 of asparagineo Disulfide bonds for stabilityo Proper folding and assemblyo Proteolytic cleavages- Biosynthesis of the Oligosaccharide Precursoro Glycosylation may: Promote proper folding Increase stability for secreted proteins Cell-cell adhesiono Preformed N-linked oligosaccharide is added to many proteins in ER- Formation of disulfide bonds occurs in ERo Protein Disulfide Isomerase (PDI) Forms and rearranges protein cysteine disulfide bonds The active site is easily interconverted between the reduced dithiol form and oxidized disulfide formo Formation of disulfide bonds (e- transfers) Substrate protein ionized cysteine thiol reacts with disulfide bond in oxidized PDI forms a disulfide bonded PDI-substrate protein intermediate Substrate protein second ionized thiol  reacts with intermediate, forming disulfide bond within substrate protein and releasing reduced PDI Regeneration  PDI sulfide bond reformed by electron transfer to reduce disulfide bond- ER monitors amounts of Unfolded Proteino Unfolded-protein response (UPR):  Presence of unfolded proteins in the rough ER increases transcription of genes that encode ER chaperones and other folding catalysts Ire1: binds excess BiP Hac1: activates transcription of genes encoding several protein-folding chaperoneso Importance of UPR: Wild-type proteins synthesized in ER cannot exit until they achieve completely folded conformation Any mutation that prevents proper folding also blocks


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