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110 Protein Sorting and TransportChapt. 10: Protein Sorting, Transport: Endoplasmic Reticulum, Golgi, LysosomesStudent learning outcomes: organelle functions• Explain sorting of proteins: free vs. bound ribosomes• Identify signals on proteins that identify destinations:Carbohydrates, amino acid sequences, lipids• Describe complexes that read signals, deliver cargo:SRP particles, Signal peptidases, chaperones, SNAREs• State steps taken for proteins destined for secretion, for a lysosome; state compartments traversed (compare nucleus); connected by vesicular transportFig 10.1 The endoplasmic reticulum (ER)1. Endoplasmic reticulum (ER); network of membrane-enclosed tubules and sacs (cisternae); extends from nuclear membrane through cytoplasm.• Membrane is continuous; largest organelle of most cells.• Rough ER is covered by ribosomes.• Transitional ER is where vesicles exit to Golgi apparatus.• Smooth ER no ribosomes, lipid metabolismFig. 10.1Fig 10.2 The secretory pathwayER and protein processing, secretion:• Pancreatic acinar cells secrete digestive enzymes• Newly synthesized proteins briefly labeled with radioisotopes, chased with nonradioactive • Proteins located by autoradiography.Fig. 10.2Fig 10.3** Overview of protein sorting**Secretory pathway: • rough ER → Golgi → secretory vesicles → cell exteriorNon-secreted proteins: • free ribosomes → nucleus, mitochondria, peroxisome**Fig. 10.32The Endoplasmic ReticulumRibosomes targeted to ER by signal sequence at NH2-terminus of polypeptide• Protein synthesis starts on free ribosomesCotranslational translocation: • Proteins translocated into ER during synthesis on membrane-bound ribosomes.Posttranslational translocation: • proteins are translocated into ER after translation completed on free ribosomes; Hsp70 Chaperone proteinsFigs. 8.22,27Fig 10.5 Incorporation of secretory proteins into microsomesEvidence for signal sequences for ER:Secretory proteins translated from mRNAs on free ribosomes are larger than the secreted proteins.With microsomes, growing polypeptide chains incorporated into microsomes, signal sequences removed by proteolytic cleavage.SDS-PAG analysis of proteins synthesized in vitrovs. secreted (s)Fig. 10.5Fig 10.6 The signal sequence of growth hormoneSignal sequences (about 20 amino acids):• stretch of hydrophobic residues• usually located at NH2-terminus of polypeptide chain.Fig. 10.6 Signal sequence of growth hormoneFig10.8 Cotranslational targeting of secretory proteins to ERFig. 10.7,8 cotranslationalCotranslational translocation:• Signal sequences are bound by signal recognition particle (SRP).• SRPs have 6 polypeptides, small cytoplasmic RNA (SRP RNA).SRP binds ribosome and signal sequence, binds SRP receptor on ER to make RER• Ribosome binds translocon; • Signal sequence insertsinto translocon• Signal peptidase releases polypeptide in ER lumen3Fig 10.9 Posttranslational translocation of proteins into ERPosttranslation translocation (more common in yeast):• Proteins synthesized on free ribosomes • Signal sequences recognized by receptors on translocon (not need SRP)• Hsp70 chaperones keep polypeptide chains unfoldedso can enter translocon• Hsp70 chaperone in ER (BiP) acts as ratchet to pull polypeptide chain throughFig. 10.9PosttranslationaltranslocationThe Endoplasmic ReticulumFig. 10.10Transmembrane (integral) proteins:• Proteins destined for incorporation into membranes initially insert into ER membrane, not release into lumen.• Transported along secretory pathway as membrane components rather than soluble proteins• Membrane-spanning regions of integral membrane proteins usually α helical regions with ~20-25 hydrophobic amino acids.• Orientations vary — N or C terminus on cytosolic side• Some multiple membrane-spanning regions.Fig 10.11 Topology of the secretory pathway** Topology:Lumens of ER and Golgi are topologically equivalentto exterior of cell. Domains of plasma membrane proteins exposed on cell surface= regions of polypeptidetranslocated into ER lumen.Fig. 10.11Fig 10.12 Insertion of membrane protein with cleavable signal sequence, single stop-transfer sequenceSimplest method for Integral protein insertion:• Signal sequence cleaved by signal peptidase during translocation, leaves NH2-terminus in ER lumen.• Translocation halts at stop-transfer sequence; protein exits laterally and anchors in ER membrane.Fig. 10.124Fig 10.13 Insertion of membrane proteins with internal noncleavable signal sequences Proteins also anchor in ER membrane by internal signal sequences not cleaved by signal peptidase.• These sequences aretransmembrane α helices • Exit translocon, anchor proteins in ER membrane, in either orientation.Fig. 10.13Fig 10.14 Insertion of protein that spans membrane multiple timesProteins that span the membrane multiple times are inserted by alternating series of internal signal sequences, transmembrane stop-transfer sequences.Fig. 10.14Fig 10.15 Protein folding in the ER• Protein folding and processing occur either duringtranslocation across ER or within ER lumen.• Lumenal ER proteins assist folding and assembly of translocated polypeptides• Hsp70 chaperone BiP binds to unfolded polypeptide chain as it crosses membrane, helps fold and assemble complexes• Disulfide (S—S) bond formation facilitated by PDI (protein disulfide isomerase).Fig. 10.15The Endoplasmic ReticulumGlycosylation:• (N-linked glycosylation) occurs on specific asparagine (Asn) residues as protein translocates into ER.• Oligosaccharide is synthesized on lipid (dolichol) carrier.• Glycosylation preventsprotein aggregation in ER, providessignals for sorting.Fig. 10.165Fig 10.17 Addition of GPI anchorsGlycosylphosphatidylinositol (GPI) anchors• Glycolipids attach some proteins to plasma membraneGPI anchors assemble in ER membrane, add to C-terminal Asn• GPI-anchored proteins aretransported as membranecomponents via secretory path. • Topology within ER dictatesthey are exposed outside of cell.Fig. 10.17Fig. 8.36GPI anchor Thy-1Fig 10.18 Glycoprotein folding by calreticulinChaperones and sensors in ER identify misfoldedproteins, divert them to degradation pathway.• Ex. chaperone calreticulin assists glycoproteins folding• Protein folding sensor passes correctly folded glycoproteins on to transitional ER. Fig. 10.18The Endoplasmic ReticulumUnfolded protein response (UPR)BiP plays role as sensor of general state of


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RU BL 424 - Lecture Notes

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