Lecture 12 - Secretory Pathway Flashcards
What Happens to Proteins in the ER lumen?
Lots of post-translational modifications
Specific proteolytic cleavages e.g. removal of signal seq by signal peptide peptidases
Glycosylation – covalent addition & processing of oligosaccharides adding glycan/sugars/carbohydrates to protein itself - this is on the lumen, tends not to find glycosylation on proteins in cytoplasm - seen on secreted/extracellular proteins
Disulphide bond formation catalysed by protein disulphide isomerase secreted proteins, transmembrane proteins on extracellular side - again, formed in lumen not generally in cytoplasm/cytosolic side
Folding & assembly of multi-subunit proteins – requires chaperones
Describe the action of chaperones on proteins in the ER lumen
Correct folding – entry is in unfolded form
in vitro many denatured proteins can refold in hours – in the ER, proteins generally reach their correct conformation in minutes, mediated by chaperones
chaperones undergo cycles of binding & release of polypeptide substrate
domains move powered by ATP hydrolysis
binding & release helps substrate polypeptide adopt correct conformation
Describe disulphide bond formation in the ER lumen
Disulphide bond formation – part of the correct folding process
The lumen of the secretory organelles is oxidising – S-S bonds form as opposed to the cytosol where free SH groups are present. Protein disulphide isomerase (PDI) make sure incorrect S-S bonds are remodelled
Lumen of the ER is oxidising environment, cytosol is reducing environment
Reduced substrate in the ER lumen - oxidised PDI reduces itself to reduced PDI and oxidises the protein forming the disulfide bonds on the protein in the process
Describe proline isomerisation
The peptide bond preceding a proline residue can adopt either the cis or trans state, and interconversion can be accelerated by peptidylpropyl isomerases (PPI) enzymes
Unique structure of proline allows it to have trans and cis conformation - most proteins, the peptide bond will be in the trans configuration - 80% of proline will be in this configuration - PPI enzymes can accelerate the conversion - this will change structure of the protein, e.g. involved in regulation of ion channels
Describe glycosylation
Lumenal Asn-Xaa-Ser/Thr sequences obtain a sugar chain (aka glycan) containing N-acetyl-glucosamine (red), mannose (blue) and glucose (green) sugars.
N-linked Glycosylation
N-glycosylation is used to check on the folding of proteins.
Unfolded proteins retain a glucose that binds to ER chaperones, calnexin and calreticulin, retaining them in the ER
Folded proteins have glucose removed and are allowed exit from the ER
What is the unfolded protein response?
The unfolded protein response (UPR) is triggered by ER chaperones when unfolded proteins accumulate in the ER.
The cause of UPR is often the expression of a mutant protein causing disease (cystic fibrosis, neurodegeneration)
UPR triggers upregulation of chaperone expression, increased export from the ER and proliferation of the ER. A prolonged UPR leads to cell death.
If folding fails, proteins are retro-translocated into the cytosol and degraded by the proteasome
Describe how proteins leave the ER
in COPII-Coated Transport Vesicles
transport vesicles form
lumen of vesicles can contain proteins which enter by bulk flow or proteins which bind to cargo receptors
proteins that bind cargo receptors have an exit signal (cellular postcode)
Assembly is initiated by Sec12 binding to Sar1, where Sar1 exchanges its GDP for GTP.
- GTP bound Sar1 binds to the ER membrane and acts as a nucleus for Sec23/24 binding. (inner COPII coat). Sec 24 binds transmembrane cargo.
- The coat assembly is completed by the binding of Sec 13/31 to the Sar1-GTP,Sec23/24 complex (outer COPII coat).
Describe how proteins move from the ER to the Golgi
Vesicles move along microtubules to carry proteins from the ER to Golgi apparatus
Form vesicular tubular cluster - ER-Golgi intermediate - deliver material to the cis Golgi
Describe ER resident protein retrieval
(retrograde transport via COP-I)
ER resident proteins get carried forward towards the Golgi along with secretory proteins. They are retrieved since they contain a ‘KDEL’ sequence.
KDEL receptors bind cargo with KDEL sequences.
KDEL receptors have a ER retreival signal which allows them to be packaged into COP-I coats (retrograde transport)
Some ER resident proteins get trapped in the vesicle by bulk flow
Don’t want disulphide bonds to be made in any old vesicle in the Golgi
COPI coat binds ER resident proteins and returns them to the ER. This involves KDEL sequences and receptors. ER resident proteins have a KDEL sequence and bind to KDEL receptor, which then binds to COPI coat
Describe glycosylation reactions in the Golgi
Different glycosylation reactions take place in each membrane of the lumen of the Golgi
Vesicular movement and budding through the Golgi stack from the cis Golgi through all the cisternae to the trans Golgi. Carbohydrate trimming and addition
What are the functional roles of glycans?
folding in the ER
stability/protease resistance of proteins
molecular interactions/recognition
immune response
Signalling
architecture of the extracellular matrix and cell-to-matrix attachment
glycan structures are altered in diseases: e.g. cancer, inflammation
Describe mannose-6-phosphate (M6P) based targeting towards the lysosome
Delivery of lysosomal enzymes
3 main points: phosphorylation of mannose, a specific M6P receptor cycling between late endosomes and the TGN, acidic pH of late endosome releases M6P from receptor
Defects in this pathway cause defects in lysosomal degradation - Lysosomal Storage Diseases have mostly neurological symptoms
pH in endosome is lower, which causes the hydrolase with its mannose-6-phosphate and its receptor to dissociate, free hydrolase trafficks to lysosome
Receptor uses retromer coat to be retrieved and go back to the Golgi to pick up more hydrolase
