Unformatted text preview: Secretory protein translocation and modification in the endoplasmic reticulum: Isolation of rough and smooth ER membranes- The rough ER is studded with ribosomes, hence its ‘rough’ appearance The smooth ER lacks ribosomes; it is a specialized region where lipid synthesis and protein sorting occur Biochemical reconstitution of ER translocation: protein targeting and removal of a signal sequenceThe signal sequence hypothesis: Günter Blobel Nobel Prize 1999 Signal sequences that target proteins for translocation across the ER membrane- N-terminal signal sequences
hydrophilic charged apolar 8-20 hydrophobic hydrophilic charged There must be great plasticity in their recognition because even random hydrophobic peptides will suffice… Helix-disrupting (Gly or Pro) at -1 & -3 The Signal Recognition Particle: a ribonucleoprotein complexPurified by rescuing the translocation activity of salt-stripped microsomes The methionine ‘bristle’ domain is flexible and can accommodate many different signal sequences A ‘hydrophobic cleft’ The Signal Recognition Particle halts protein translation upon binding the signal sequence of a nascent chain coming out of the ribosome Signal sequence recognition Translation is paused by interfering with access of translation elongation factors Targeting of nascent chain/ribosome complex to a receptor in the ER membrane- SRP contacts the signal sequence and the ribosome, and halts translation. The SRP/SRP-receptor complex hands-off the nascent chain to the translocon, which opens upon signal and ribosome binding Docking of SRP-ribosome complex to SRP receptor diffusion ‘Kinetic proofreading’ by SRP and its GTPasesGTPases functions as ‘timing devices’ GEF: guanine exchange factor GAP: GTPase activating protein GTP/GDP ratio in cells is 10:1 Ribosome is a GEF for SRP signal binding inhibits SRP p54 GTPase & translation The GDP-bound forms of SR & SRP do not interact Translocon is GEF for SR αβ After docking: SR is a GAP for SRP/SRP is a GAP for SR mRNA’s are tethered to the ER membrane via polyribosomes that re-cycle: Cryo-EM analysis of the ribosome/translocon complexPurified ribosomes and Sec61p complex were mixed and processed for cryo-EM The hole in the ribosome where the nascent chain comes out aligns perfectly with the central opening in the bound Sec61 complex. Dynamic structure and regulation of the Sec61 translocon complex and its “double-plug’ system
The “double-plug’ system Sec61 αβγ translocon complex double-plug A four translocon complex? Probing the nascent chain environment in the ribosome-translocon channel: Is it aqueous?
Collisional quenching: of fluorophore
Ribosome-arrested Nascent chain complex Tight seal fluorophore Its fluorescence is environmentally sensitive Test diameter of the channel using collision probes of different sizes Two modes of protein translocation across the ER membrane: co-translational versus post-translational translocation stuffing stuffing trapping or pulling -plasma membrane plasma membrane Sec71 and Sec72 are signal sequence receptors > 90 % of secretory proteins in eukaryotes Energy for directional translocation is provided by the ribosome… Require cytosolic chaperones & molecular ratchets DNA J domain in Sec63p enhances ATPase activity of BiP and the energetics of protein translocation into ER… Translocation of a soluble ER lumenal protein across the ER membrane (the signal must be removed)A binding site for the signal sequence inside the translocon triggers it open; the signal also contacts lipids Need signal sequence and flanking signal to serve as substrate for the signal peptidase Lateral opening of the translocon (ribosome not shown) A plug or peculiar structural conformation of the translocon keeps it sealed Translocation of a single-pass transmembrane protein across the ER membrane- Translocation of a protein with an internal, non-cleaved signal sequence• The orientation of insertion of the start -transfer signal (signal sequence) determines the membrane topology of the protein • Positive charges flanking the signal sequence determine the orientation of insertion Type I The established topology of proteins in the ER is maintained across the organelles the secretory pathway Type II Translocation of a multi-pass transmembrane protein across the ER membrane- Modification to proteins upon arrival into the lumen of the ERER environment specialized for folding of membrane and secreted proteins
A. Signal peptide cleavage B. N-glycosylation C. Oxidizing S-S bond formation D. Folding enzymes/quality control Protein disulfide isomerase (PDI) Peptidyl prolyl isomerase Chaperones HSP70 (BiP) ER57 GRP94 Calnexin calreticulin Glycosylation of nascent proteins in the ERGlycosylation occurs co-translationally NxS/T is a glycosylation signal Most proteins residing in secretory organelles and the plasma membrane are glycoproteins Protein folding in the ER: Formation of disulfide bonds aided by protein disulfide isomerase
Glutathione in cytosol
γ-Glu—Cys—Gly γ-Glu—Cys—Gly + NADP + + NADPH + H + S Glutathione reductase γ-Glu—Cys—Gly Oxidized (GSSG) Reduced (GSH) S ER GSH = 2 GSSG GSH = 60 GSSG Protein Folding in the ER: The Role of Calnexin/CalreticulinCycles of Binding and Release by Calnexin/Calreticulin Help Proteins Fold Correctly Mostly glucosyl transferase determines when a protein is folded properly or not. ER-dislocation of unfolded proteins (ER lumenal or membrane) Unfolded proteins that have lost a terminal mannose residue are discarded as junk… Correct protein folding in the ER lumen is essential for human health- The Unfolded Protein Response in the ER eliminates unwanted proteins
Protein misfolding can be caused naturally by a mutation in a protein, or by an increase in temperature. Experimentally it can be induced by chemical inhibitors of glycosylation (tunicamycin) or by reducing agents (beta-mercaptoethanol) Hsp70’s, PDI, calreticulin, etc The Unfolded Protein Response in the endoplasmic reticulum via Ire1- ...
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