MCB L11-12
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MCB L11-12 - Marcador
MCB L11-12 - Detalles
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| Co-translational targeting | An N-terminal signal peptide directs the nascent protein into the ER lumen: and from there other parts of the secretory pathway can be accessed. |
| Protein secretion | 1. From the cytosol, proteins cross (translocate) the ER membrane and exit the ER in vesicles. 2. These vesicles then fuse with the Golgi, and the proteins are transported through the Golgi into secretory vesicles. 3. After fusion of the last vesicles with the plasma membrane, the protein content (cargo) is secreted to the outside of the cell. |
| Entry into the ER requires a signal peptide | MRNAs that encode secreted proteins encode a 5’ signal sequence (SS) fused in frame to the sequence encoding the mature secretory protein: this signal sequence encodes an N-terminal signal peptide (SP). It seems that SPs are free to evolve rapidly, as long as they retain their overall features: a +ve charge towards the N’ terminus and a hydrophobic stretch. Do not bind the ER membrane directly: because the SP is recognised by signal recognition particle (SRP) in the cytosol – and this targets the SP to the translocon. |
| The SRP cycle | 1.An emerging ER signal peptide 2.is captured by SRP (recognition step) 3.which directs the SRP/SP complex (targeting step) to the α subunit of SRP receptors in the ER membrane 4.allowing recruitment of a closed translocon 5.which opens to allow entry of the SP as a loop whilst the SRP/SRP receptor complex is dismantled and SRP is recycled. 6.Signal peptidase removes the SP, which is released into the ER membrane. There are millions of active translocons in the ER membrane, so at any one time there could be millions of cleaved signal peptides – which could disrupt the ER membrane. |
| The specificity of ER targeting | Depends upon: broad substrate specificity in the recognition step and the folding of the SP into a loop by SRP, which allows signal peptidase to cleave the signal within the translocon releasing the SP into the ER membrane, which causes problems downstream, because SPs disrupts the ER membrane, requiring theirremoval by chopping with signal peptide peptidase |
| What happens in the ER lumen? | The ER lumen is a major site of protein folding: this requires ER chaperones. Two major ER modifications: Folding proteins may become N-glycosylated and/or disulfide-bonded depending on whether they have the right amino acid sequence motifs for these events. If a protein does not fold properly, it will fail a quality control check carried out by ER chaperones. Misfolded proteins are ejected from the ER in a process called retro-translocation, and are then degraded in the cytosol. |
| N-glycosylation of proteins | 1. N-glycans act as flags for folding and ER Quality Control. The removal of two Glc residues from the core N-oligosaccharide in the ER allows interactions with ER chaperones required for efficient folding of N-glycosylated proteins. Removing the protein from the folding environment and signalling that the protein is now deemed fit to be passed to the Golgi. 2. A core N-glycan is very large and made of hydrophilic sugars. N-glycans therefore increase protein solubility and can reduce aggregation problems during folding in the ER. 3. N-glycans are bulky and therefore constrain the α-carbon backbone of the polypeptide: they therefore influence folding rates and final protein conformation. |
| Disulfide bond formation | Disulfide bonds do not form readily in reducing conditions (the cytosol): they require oxidising conditions (the ER in eukaryotes or the bacterial periplasm). They form where 2 cysteine residues are brought close together during protein folding. The biological catalysts are protein disulfide isomerase (PDI) and its relatives. PDIs can make, break and shuffle disulfide bonds. Covalent S-S bonds contribute to the stability of protein tertiary structures (of SECRETED proteins, not cytosolic proteins). Many are essential for the activity of proteins. |
| PDI can isomerise (shuffle) disulfide bonds, join and separate two proteins | PDI recognises unstable proteins, binds as a chaperone, and forms a mixed disulfide bond. Isomerisation of disulfide bonds continues until the client protein reaches a stable conformation, when it is released from PDI. |
| ER Protein folding processes do not work in isolation – they are co-ordinated: e.g., MHC Class I assembly | 1.BiP maintains solubility of MHC Class I HC until β2 microglobulin binds. 2. and N-glycosylation allows entry into a folding environment 3. provided by the chaperone calreticulin 4. and its PDI-related partner ERp57 5. which is disulfide-linked to tapasin6. This allows assembly and folding of Class I molecules, providing a groove for insertion of a peptide 7. fed in from the cytosol by the TAP transporter that is recruited by tapasin 8. The peptides are derived from proteosomal degradation. |
| What happens to an ER protein that fails quality control? e.g., an MHC Class I HC that has failed to find β2m | 1. If the MHC Class I HC has a long residence time with ER chaperones 2. this allows access of a mannosidase that removes one particular mannose 3. removing this protein from the folding environment. 4. The protein is unfolded and fed through a multisubunit DISLOCON 5. and is ubiquitylated during dislocation, which targets it to the proteasome 6. where it is deubiquitylated and degraded. |
| Other secretory system modifications | 1. Attachment of sugars to oxygen atoms of amino acids (O-glycosylation). 2. Proteolytic cleavage, e.g. to activate a protein such as albumin 3. Addition of lipids to permit/maintain membrane targeting. |
| Attachment of sugars to oxygen atoms of amino acids | O-linked glycosylation occurs in the ER/Golgi apparatus in eukaryotes. It also occurs in Archaea and Bacteria. O-N-acetylgalactosamine (O-GalNAc) may be attached to serine or threonine residues - the high concentration of carbohydrates gives mucus its "slimy" feel. O-mannose can be attached to serine and threonine residues in secretory pathway proteins. O-mannosylation is common to both prokaryotes and eukaryotes. O-fucose and O-glucose can be added to cysteine residues. |
| ER selective transport: five major interdependent strategies | Cargo capture: receptor-mediated export of proteins from the ER to the Golgi complex in coatamer protein II (COPII) vesicles. Bulk flow: some proteins and lipids are included in COPII vesicles by default Retention: prevents proteins from entering the transport vesicles Retrieval: retrograde transport from the ER-Golgi intermediate compartment (ERGIC) /early Golgi back to the ER ERAD: cytosolic elimination of ER proteins that fail quality control |
| Cargo capture and anterograde transport | In the ER, secretory cargo is loaded into COPII transport vesicles at ER exit sites (ERES). This requires export signals in fully folded client proteins and cargo receptor proteins in the vesicle membrane. COPII vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC). When COPII vesicles are close to the cis-Golgi membrane, they shed their coats - COPII components are recycled for use in other vesicles. The receptors usually return to the ER by retrieval pathways. |
| Bulk flow and retention | Export by bulk flow does not require receptors or export signals. Some soluble and membrane proteins enter COPII vesicles by default. There is a biotechnological use: foreign proteins directed into the ER are often secreted into the growth medium as soluble proteins that are relatively easy to purify. Retention: some proteins are selectively excluded from COPII vesicles. |
| Retention: some proteins are selectively excluded from COPII vesicles. | For retrograde transport from ERGIC and the cis-Golgi, COPI coated vesicles retrieve transport machinery, cargo receptors, lipid membrane, and escaped ER-resident proteins. Retrieved membrane proteins typically possess a C-terminal dilysine motif or a close variant. Retrieved soluble proteins typically have a C-terminal ‘KDEL’ motif. |
| Retrieval and retrograde transport (Rab6) | There is also a retrograde route governed by the small GTPase Rab6 protein: these tubular elements are independent of COPI. Not currently well-characterised. |
| Further destinations and ERAD | Trans Golgi network (TGN), late endosomes and lysosomes: requires a mannose-phosphate signal. TGN and early/sorting endosomes TGN, secretory vesicles and exterior ERAD. Cytosolic destruction of misfolded proteins in the ER. There is some evidence that ERAD substrates cycle between the ER and the Golgi. |
| Some stresses that increase misfolding/stimulate unfolding | Abiotic stresses: Heat stress, Osmotic stress and High light intensity. Biotic stresses: Infection and Stress related hormones. |
| The unfolded protein response (UPR): the ER as a stress sensor | The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signalling pathways - the unfolded protein response (UPR). UPR sensors monitor the ER folding status of proteins in the ER. Following sensing, the UPR adjusts the folding capacity of the ER according to need |
| There are multiple sensing mechanisms, mediated by | Ire1 (Inositol-requiring Enzyme 1), PERK (PRKR-like endoplasmic reticulum kinase) and ATF6 activating transcription factor 6) |
| Ire1 uses an unusual splicing mechanism | Most eukaryotic splicing requires two transesterifications coordinated by snRPs, and occurs in the nucleus. By contrast, Ire1 in plants, mammals and fungi has RNAse activity that recognisees a specific cytosolic RNA, and which removes a highly conserved intron. The exons are fused using an RNA ligase activity. |
| How do we know this? Measuring ER stress | HAC1p (yeast), XBP1s (mammals) and bZIP60 (plant) proteins are all transactivators: once expressed from spliced mRNA, they enter the nucleus and stimulate expression of UPR genes. Responses: Increased ERAD (ER-associated protein degradation) Increased chaperone production (improved protein folding) Increased protein trafficking rates (clearing misfolded proteins from the ER, particularly in yeast) |
| Ire1 also has a general RNase activity (RIDD) and can also stimulate signalling via c-Jun N-terminal kinase (JNK). | Transcription factor- to nucleus: Increase ERAD, folding and trafficking. Regulated IRE1 dependent decay of mRNA (RIDD): Regulated IRE1 dependent decay of mRNA (RIDD). RIDD degrades mRNA in complex with ER-associated ribosomes. |
| PERK is also a stress sensor. PERK vs IRE1 | Share similarities: The ER domains of PERK and IRE1 share sequence and structural similarity (from yeast and mammals). The cytosolic portion of PERK and IRE1 both possess kinase domains that autophosphorylate in trans. Display differences: IRE1 activation leads to specific splicing and production of transactivators of transcription. However, PERK activation leads to interference with translation. |
| PERK: likely mechanism | Same as IRE1 but activates elf2a = reduces translation globally and this reduces protein expression and relieves stress. If ATF4 translation promoted, CHOP is expressed, apoptosis occurs |
| ATF6: likely mechanism | With ER stress is activated and goes from ER to Golgi. It is then cleaved, ATF6f is a TF to nucleus and increase: ERAD, ER chaperones, Bip and other. |
| Ire1, PERK and ATF6: other models | Competition model: Here Ire1, PERK and ATF6 are the direct sensors of ER stress sensors. Bip binds the cytosolic domains. Unfolded proteins compete for BiP. Direct association model: Here, misfolded proteins bind directly to the sensors, stimulating activation and leading to UPR responses. BiP maintains solubility of inactive Ire1, PERK and ATF6 in this model. Allosteric model: Bip binds the sensors through a different site than the unfolded protein binding site: binding misfolded proteins induces a conformational change so Bip releases the sensors. Here BiP is regarded as the direct sensor of ER stress. |