Lecture 14. Protein Sorting II 2024 PDF

Protein Sorting II How proteins enter the endomembrane system ECB6 Chapter 15 pp. 526-540 MBoC7 Chapter 12 pp. 698-723 The ER is the sole “gateway” to the endomembrane system The ER is an extensive network of flattened sacs and branching tubules Tubule netwo...

Protein Sorting II How proteins enter the endomembrane system ECB6 Chapter 15 pp. 526-540 MBoC7 Chapter 12 pp. 698-723 The ER is the sole “gateway” to the endomembrane system The ER is an extensive network of flattened sacs and branching tubules Tubule network is pulled to the edges of a cell Network is densest around the nucleus  The membrane layers are interconnected Lumen of the ER The nuclear envelope is part of the ER, the lumen is continuous sheets tubules Rough ER: Flattened sheets Coated with ribosomes Smooth ER: Tubular Less abundant in most cell types  Rough ER is defined by its ribosome coating Most abundant near the nucleus but found throughout the cell Site where proteins are synthesized, folded, and glycosylated The size of ER is controlled by the demand for protein  Smooth ER: more abundant near cell edges and expanded in certain cell types Most lipids and cholesterol are synthesized in smooth ER Site for intracellular Ca2+ storage Site of “exit” of transport vesicles carrying newly synthesized proteins and lipids to the Golgi apparatus Expanded in liver, muscle, endocrine cells that synthesize steroid hormones  ER match up Choose rER or sER a) Synthesis of phospholipids and cholesterol b) Synthesis of secreted proteins and proteins of the endomembrane system tubules c) Protein folding and modifications: glycosylation, addition of disulphide bonds d) Synthesis of steroid hormones e) Detoxification (in liver) of hydrophobic drugs, toxins (e.g. ethanol) to H2O-soluble forms for secretion f) Storage of intracellular Ca2+ g) Exit sites for vesicle trafficking Most proteins entering the ER use a co-translational import mechanism Proteins enter the endoplasmic reticulum while being synthesized = co-translational import Polyribosomes How ribosomes are docked at translocation channels in the ER membrane Docking relies on a signal sequence containing 6-15 hydrophobic amino acids, usually at the protein N-terminus. This signal sequence is often called a “start transfer sequence” because it starts the transfer of the polypeptide into the ER Soluble proteins are synthesized through a channel into the lumen of the ER Integral membrane proteins are partly translocated and become embedded in the membrane Using genetic engineering techniques, you have created a set of proteins that contain two (and only two) conflicting signal sequences that specify different compartments. Predict which signal would win out for the following combinations. Why? A.Signals for import into the nucleus and import into the ER B. Signals for import into the mitochondria and import into the ER The ER import signal is recognized by a co-receptor that docks the ribosome at translocators in the ER membrane 3 1 4 2 1. As soon as the ER import signal is exposed, it is bound by a signal recognition particle 2. Translation pauses while the SRP brings the whole complex to an SRP receptor associated with a translocation channel in the ER membrane 3. Docking of the ribosome on the translocator displaces SRP – translation resumes Signal Recognition Particle (SRP) is an ancient ribonucleoprotein co-receptor that recognizes ER import signals The binding pocket is rich in methionine, accommodates hydrophobic ER import signals GTP hydrolysis by SRP and SRP receptor allows SRP to release from the SRP receptor when cargo is delivered and change back to its original shape SRP in eukaryotes = six polypeptides + one RNA molecule Conserved in prokaryotes, the system arose early in evolution SRP has a hinge-like mechanism Binding to the ER import signal sequence triggers a conformation change that exposes a binding site for SRP receptor The pause domain binds to the interface between the large and small ribosome subunits, blocking elongation factor from binding, translation is paused Entire complex docks with SRP receptor on ER membrane  SRP serves as a “molecular matchmaker” ER import signal is recognized twice SRP binding pauses translation so that peptide synthesis does not complete in the cytosol Proper docking on the ribosome results in GTP cleavage, release of SRP and SRP receptor from the translocation channel The translocation channel stays closed until the ribosome is properly docked so the permeability barrier of the ER membrane is maintained at all times Signal sequence binding opens the translocation channel by displacing a loop that acts as a “plug” The pore has four identical complexes: Pore open sideways at seam: -diffusion of cleaved signal sequence or -release of transmembrane proteins into the bilayer How a soluble protein crosses the ER membrane into the lumen 3. Signal is 4. Signal cleaved and peptide is released. Pore rapidly closes. degraded 1. Binding of 2. Signal sequence signal sequence stays bound to pore opens pore during import Watch Movie 15.4 Sometimes, the start-transfer signal sequence is not at the N- terminus of the protein. What then? The start transfer sequence binds in the pore with its most basic (+) end up (towards the cytosol). The basic end is typically toward the N-terminus. These internal signal sequences are not cleaved – they form a membrane- spanning domain that anchors the protein in the bilayer. Pairs of start and stop transfer signals determine how transmembrane proteins integrate into the lipid bilayer Single pass transmembrane protein  Pairs of start and stop transfer signals determine how transmembrane proteins integrate into the lipid bilayer Multi pass transmembrane protein Match the membrane protein with its sorting signals Assume that the basic part of all internal start-transfer sequences Is towards the N-terminus  Translocated polypeptide chains are folded and glycosylated in the lumen of the rough ER cytosol lumen Chaperones (folding) glycosyl transferase (adds sugars) Enzymes that add lipid anchors to protein disulphide proteins isomerase (makes disulphide bonds) Most proteins entering the ER are in transit to other parts of the endomembrane system or cell surface Resident proteins of the ER require an ER retention signal “KDEL” to stay put  Modification of proteins in the rough ER GPI anchors are added Disulphide bridges are formed Lipid-linked integral membrane proteins Most proteins synthesized in the rough ER are glycosylated by the addition of a common N-linked complex oligosaccharide N N-glycosylation can promote the folding or activity of certain proteins N-glycosylation is more generally used as a marker of protein folding The role of N-lnked glycosylation in ER protein folding ER chaperones called calnexin and calmodulin bind to N- glycosylations with a single terminal glucose = signal for “unfolded protein” Trimming off the terminal glucose releases the protein from calnexin for a round of folding. If folding is incomplete, an enzyme adds back the terminal glucose, restoring its affinity for calnexin. If unfolded for too long, a slow- acting mannosidase removes mannoses so glucose cannot be added back, and the protein is routed for degradation The export and degradation of misfolded ER proteins N-glycosylation is used as a tag to mark the state of protein folding Only properly folded proteins are allowed to leave the ER Up to 30% of proteins are estimated to never exit the ER and get destroyed because of the stringency of this monitoring system.  Defects in the stringency of the ER quality control mechanism are a leading cause of cystic fibrosis disease Class II mutations disrupt folding of the Cl- channel triggering an unfolded protein response in the ER Research shows that many such mutant channels have partial function, so correctors are being developed that block unfolded protein response. Lumacaftor is an example of a corrector that acts as a chaperone during protein folding to increase the number of channels that reach the plasma membrane Thanks to our understanding of unfolded protein response and new treatments median age now 40+ for people cystic fibrosis Read more: https://journals.sagepub.com/doi/10.1177/1753465815601934 Lipids and proteins exiting the ER move next to the Golgi complex for further processing Vesicle transport is the method of transportation within the endomembrane system Vesicle Transport: ER to Golgi ECB6 Chapter 15 pp. 532-544 MBoC7 Chapter 13 pp. 749-777