Lecture 12 - Protein Sorting: Endoplasmic Reticulum PDF

Protein sorting: Endoplasmic Reticulum BIOL2020 Prof. Nicanor González Chapter 12 Nearly all proteins, except a few inside mitochondria and plastids, begin their synthesis on ribosomes in the cytosol. Finds a messenger RNA strand and ensures that each codon pairs with the anticodon Small subunit...

Protein sorting: Endoplasmic Reticulum BIOL2020 Prof. Nicanor González Chapter 12 Nearly all proteins, except a few inside mitochondria and plastids, begin their synthesis on ribosomes in the cytosol. Finds a messenger RNA strand and ensures that each codon pairs with the anticodon Small subunit Large subunit Catalytic site (RNA) Contains the active site of the ribosome: the site that creates the new peptide bonds when proteins are synthesized. RNA molecules: orange and yellow New proteins must go from a ribosome in the cytosol to the organelle where it functions. It does so by using sorting signals in its amino acid sequence The main steps of the secretory pathway 1. 2. 3. 4. 5. 6. 7. Translates from mRNA in the ribosomes in the cytoplasm Enters the ER lumen Goes from the ER to the Golgi in a vesicle Transits the Golgi Leaves the Golgi in a vesicle The vesicle fuses the cell membrane It is outside electron microscopy give snapshots of the secretory pathway George Palade Nobel Prize 1974 RER → Golgi → Vesicles → Membrane Genetic screen for secretion defective yeast Randy Schekman overviews the secretory pathway https://www.ibiology.org/cell-biology/protein-secretion/ Mutants fall into distinct categories depending on where the mutated protein was required 1. 2. 3. 4. 5. 4 3 5 2 1 Fail ER import Fail to produce ER vesicles Vesicles don’t fuse to Golgi Fail to leave the golgi, Golgi vesicles do not form Vesicles don’t fuse with cell membrane The main proteins involved in protein trafficking were discovered in yeast, including the Sec proteins The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol the ER has a single internal space, called the ER lumen The rough ER has ribosomes bound to the membrane surface. The smooth ER lack ribosomes and is dedicated to other functions such as the biosynthesis and metabolism of lipids. Ribosomes initiate translation in the cytosol ● Pay attention to the direction! Cotranslational import into the ER is the first step in protein secretion Ramanujan Hegde https://www.ibiology.org/cell-biology/protein-localization-inside-cells/ The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which binds to the signal sequence, and an SRP receptor in the ER membrane When a signal sequence binds, SRP exposes a binding site for an SRP receptor, which is a transmembrane protein complex in the rough ER membrane. Membrane-bound ER ribosomes make proteins that are co-translocated across the ER membrane. Free ribosomes, unattached to any membrane, synthesize all other proteins The Polypeptide Chain Passes Through a Signal Sequence–gated Aqueous Channel in the Translocator (or Translocon) The core of the translocator is, called the Sec61 complex This is a Cryo EM image ! In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER ● Protein folding in the ER involve: Chaperone proteins, Disulfide bonds, and glycosylation ER MOLECULAR CHAPERONES Prevent protein misfolding and aggregation Hsp70 (aka BiP) N-Linked Glycosylation OST: oligosaccharyltransferase Disulfide bonds PDI: Protein disulfide-isomerase Disulfide bonds are a post-translational modifications that occur in the ER Cys Cys ● Protein disulfide-isomerase (PDI) forms disulfide bonds Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a Common N-Linked Oligosaccharide Oligosaccharides Are Used as Tags to Mark the State of Protein Folding folded properly: remove last glucose not folded properly: add a glucose Calnexin binds to monoglucosylated on incompletely folded proteins and retain them in the ER. Transmembrane domain Glucose binding ● ● ● ERp57 binding Calnexin is a chaperone Recruits an oxidoreductase (ERp57) to add more disulfide bonds If folding is good, GlsII removes the final glucose residue. ER Chaperones prevent protein misfolding and aggregation. e.g, Hsp70/BiP Hsp70 peptide ATP binding ● ● ● peptide binding Protects peptides from interacting with other misfolded proteins Create a folding environment give folding a second chance The Chaperone, 1893 Maximilian Wachsmuth chaperones unfold misfolded proteins to give them a 2nd chance of folding properly Misfolded proteins are recognized because they have exposed hydrophobic residues ● ● Hydrophobic residues should not be outside Folding sensors recognize Hydrophobic surfaces Summary ● ● ● ● ● Secretory pathway Proteins are forced inside the ER lumen (translocator/translocon) ER proteins help nascent peptides fold Sugars are the “properly folded” code If okey, go to the Golgi in a vesicle Cotranslational import into the ER ● Once the protein is in the ER lumen is already outside of the cell N-Linked Glycosylation N-Linked Glycosylation OST: Oligosaccharyltransferase adds 3 Glucose ▲, 9 Mannose ⚫. and 2 N-acetylglucosamine⬛ Gls: Glucosidase removes Glucose ● Glucose is a time signal to sequester proteins in ER until they are folded properly