MCB Flashcards
The secretory pathway
Importance in
- Protein modification and maturation in ER and Golgi - disulphides bonding, glycosylation, folding and assembly of quaternary structure
- Secretion of appropriate proteins go outside the cell via exocytosis - for cell to cell, cell to tissue/ organ and cell- whole organism interaction and communication. Regulatory
- internal protein quality control in Golgi, destruction in lysosomes
- removal/ usage of extracellular material via endocytosis/ phagocytosis (in endosomes and/ or lysosomes)
It is essential that proteins intended for secretion outside the cell are exported in appropriate amounts, time and at high quality, and proteins that belong within cell are not accidentally secreted out
ER targeting signal
N- terminal, approx 10-16 residues
Short gap between N terminus and start of targeting signal
At least one Arg or Lys residue (basic, positive)
Set of 6-12 hydrophobic aa residues
Long stretches of hydrophobic aa usually belong in membranes but about 20 aa required to fully span the membrane- the ER targeting signal is too short for this, so this sequence motif instead acts as a targeting signal that says “I belong in the ER”
Mechanisms for protein import into ER
Protein translation begins on free ribosome in cytosol
After about 30 reissues translated, ER targeting signal becomes exposed
Signal recognition particle (SRP) is a switch protein and a GTPase, compromised of protein and RNA
SRP recognise and binds to ER targeting signal via hydrophobic residues
SRP binding to target signal- halts translation temporarily by blocking aminoacyl tRNA entry pocket, also causes conformational change in SRP with increased affinity for SRP receptor on ER surface
Enter ribosome/ mRNA/polypeptide/SRP complex socks onto ER surface via SRP-R
Adjacent SRP is a protein transport channel called translocon
Ribosome now positioned directly over protein transport channel
GTP hydrolysis causes conformational change in SRP, reduces affinity for SRP-R so SRP is released
SRP release allows translation to resume
Targeting signal is cleaved (signal peptidase/ protease)
Translation continues and drives import process through the protein transport channel.
Translation continues until stop codon is encountered.
ER protein import summary
ER targeting is a co-translational event - after translation of initial 30 aa, mrna / ribosome/SRP complex docks directly onto protein transport channel on ER surface and translation occurs simultaneously with import (unlike nuclear and mitochondrial import, which are post translational)
Proteins are imported into ER in unfolded state (like mitochondrial import but unlike nuclear import of fully folded proteins). Must be a different reason In ER- due to protein translation driving ER import, nowhere else for protein chain to go
ER targeting signal- N-terminal face. Same as mitochondrial targeting, unlike NLS
ER targeting signal more sequence defined - Arg/Lys followed by 6-13 residues (more similar to nuclear localisation signal with its stretch of basic amino acids, and unlike mitochondrial signal which is more of a consensus structure (amphipathic alpha helix)
On important, ER targeting signal is cleaved by ER lumen protease (signal peptidase), like mitochondrial matrix, cleavage of its signal, but unlike NLS where not cleaved.
Once the ribosome is docked onto the ER, the ribosomal translation process drives protein transport across the ER membrane (unlike mitochondrial import which is driven by the proton gradient/ membrane potential)
ER modification 1: disulphide bond formation (oxidation of sulphydryl groups from 2 cysteine residues to form cystine)
Not all proteins necessarily contain Cys residues
Not all proteins that contain multiple cys resides necessarily contain any disulphide bridges
Not all Cys residues present are necessarily involved in disulphide links
But those that are must form these bonds correctly to provide native protein structure and function.
Strong covalent links
Added complications intra and inter molecular disulphide links
ER- oxidative environment (conducive to forming disulphide bonds) (cf, cytosol, nucleus and mitochondria are reducing environments) disulphide links may form improperly in ER due to - oxidative environment, - natural tendency for closest Cys residues to interact after translation.
ER has mechanisms to stop inappropriate S-S links forming: soluble ER lumen protein
Protein disulphide isomerase (PDI)
PDI facilitate protein folding (chaperone) plus formation and rearrangement of disulphide bonds. PDI releases protein when it reaches most thermodynamically stable folded conformation
ER modification 2- N linked glycosylation
Glycosylation - addition of a sugar moiety or a preformed olgiosaccharide onto a protein that goes into ER
Glycosylated protein becomes glycoprotein (covalent links, so strong)
Enzyme olgiosaccharide protein transferase (OPT) in ER lumen recognises sequence
Adds sugars to amino groups on ASN residue, hence N linked
- aids stability
- involved in cell adhesion machinery
- specific glycosylation patterns help redirect proteins to lysosome
- lubrication
- proper or enhanced function of proteins
After protein leaves ER, has 3 glucose /1 mannose trimmed- quality control step to ensure proper folding in ER before sent to Golgi or out of cell
ER modification 3: protein assembly
Coming together of multiple subunits often occurs in ER
Sometimes quality control is so good it causes problems
Retrieval of ER proteins: ER retention signal (KDEL) and KDEL receptor
ER Modification proteins are soluble and in ER lumen. Although ER resident proteins, they can easily get into vesicles and pass into the secretory pathway, so there are mechanism to retrieve them back into the ER.
They contain an extra C terminal targeting signal called the ER retention signal, the sequence of which is KDEL- Lys, asp, glu, leu
KDEL signal recognised by membrane bound KDEL receptor which binds tonER resident proteins. Different affinities of KDEL receptor for KDEL signal in two compartments caused by different pH: lower pH in Golgi, increases receptor affinity for KDEL, higher pH in ER decreases affinity, so proteins are collected in Golgi and returned to ER
Golgi apparatus
Sorting centre for proteins (can go from here, back to ER, to vesicles, plasma membrane, lysosomes or endosomes)
Proteins travel in cisternae
Further trimming and/ or addition of sugars on N glycosylated proteins
Olinked glycosylation (1-4 sugars covalently attached to Ser and Thr resides via hydroxyl groups, hence Olinked)
Lysosomes
Single membrane organelles
Contain enzymes to degrade endocytosed material
Can also degrade intercellular contents, even whole old organelles, contain 40 hydrolytic enzymes (proteases, lipases, nucleases)
These hydrolytic enzymes do the work in the lysosome, so they need to get there correctly
Synthesised in the ER, go through Golgi but recognised by Golgi as proteins that should not go outside the cell- should instead be directed to lysosomes
ER> Golgi > endosomes > lysosomes
Lysosomal enz is N- link glycosylated
In Golgi, another enzyme recognises it as lysosomal protein, so phosphorylatesna mannose sugar to mannose 6 phosphate. Recognised by M6P receptor.
If an enzyme intended to degrade targets in the lysosome is synthesised in the ER, what’s to stop it being accidentally degrading things in the ER on its way to the lysosome.
Hydrolytic enzymes must be kept inscribed until the reach lysosome- low optimal pH.
Regulated versus constitutive protein secretion
Regulated:
Some proteins are loaded into vesicles at very high concentrations of crystalline and stored
Can provide very high and rapid release of secreted protein all at once in response to signal at particular times
Constitutive:
Other proteins are secreted through vesicles at lower levels but more or less continually as they are made
Proteins destine for roles in the plasma membrane
Such as receptors, transporters, channels and pumps
Membrane proteins have characteristic long hydrophobic regions
They enter the first step of secretory process, but not released as soluble proteins into the ER lumen
When their hydrophobic regions emerge from ribosome, interior of translocon/ transporter (hydrophilic) avoids interaction with them by making conformational changes to its alpha helices
Interior of translocon opens up and spits the hydrophobic region out laterally into the adjacent ER membrane.
Once in ER membrane, they bud off as vesicles to pass through Golgi and secretory vesicles stages, eventually fuse with plasma membrane
Clathrin
Bud off membrane bound vesicles from membrane systems and fusion of these vesicles with other membrane systems
Occurs in receptor mediated endocytosis
