Lecture 11. ER Events Flashcards
What is ER targeting?
Starts in the cytosol
The ER is NOT a major site of protein manufacturing, the cytosol is
ER targeting requires proteins to be made in the cytosol initially and is a later step
What happens in co-translational targeting?
Proteins are made but the first part of the protein is extruded (N-terminus)
N-terminal acts as a signal peptide and directs the nascent protein into the ER lumen (engages with translocon): and from there other parts of the secretory pathway can be accessed
What happens in the secretory pathway during protein secretion?
- From the cytosol, proteins cross (translocate) the ER membrane and exit the ER in vesicles
- These vesicles then fuse with the Golgi, and the proteins are transported through the Golgi into secretory vesicles (ultimately fuse with the outside of the cell)
- After fusion of the last vesicles with the plasma membrane, the protein content (cargo) is secreted to the outside of the cell
What happens to some secretory pathway proteins that are directed from the Golgi that don’t go to the plasma membrane?
Some secretory pathway proteins are nucleus directed from the Golgi to the late endosomes/lysosomes, and some are retained in membranes
What is the entry point for the secretory pathway?
The endoplasmic reticulum (ER)
What does entry into the ER require?
A signal peptide (part of the primary sequence of the protein)
mRNAs that encode secretory pathway proteins encode a 5’ signal sequence (SS) in frame to the sequence encoding the mature secretory protein: this signal sequence encodes an N-terminal signal peptide (SP)
SP removed by proteolytic cleavage to provide mature protein
What do ER signal peptides look like?
Don’t all look the same
Variable length of ~15-30 amino acids
All SPs can be recognised due to overall similarities between them
What do all ER signal peptides tend to have?
A +ve amino acid (Lys, Arg) towards the N’ terminus and a hydrophobic stretch that is recognised by signal recognition particle (SRP). The cleavage site usually follows a small amino acid
No sequence specificity for a single peptide
How does a SP ensure that the translating ribosome binds the ER membrane?
Because the SP is recognised by signal recognition particle ( SRP) in the cytosol – and this targets the SP to the translocon
How does a protein enter into the ER?
An emerging SP captures SRP (recognition step) and binds the α subunit of an SRP receptor (targeting step), recruiting a closed translocon (protein-conducting channel)
Translocon opens, allowing entry of the SP into the channel. The SRP cycle SRP/SRP receptor complex is dismantled and SRP is recycled
Signal peptidase (SPase) cleaves the SP from the protein, and the SP leaves the translocon through a lateral gate
Signal peptide peptidase (SPPase) cleaves the SP into two, and translation continues until termination
What influences the specificity of ER targeting?
Depends upon broad substrate specificity in the recognition step
The folding of the SP into a hook shape by the 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 their removal by chopping with signal peptide peptidase (although Saccharomyces lacks SPP)
How are ER targeted nascent proteins formed?
Formed in locally crowded conditions
Nascent membrane-bound polyribosomes tend to crowd on ER lumen, so ER chaperons are required for efficient folding to allow the proteins to enter the ER
What are the two major ER modifications that can happen to folding proteins?
Folding proteins may become N-glycosylated and/or disulphide-bonded (only forms between two adjacent cysteines in oxidising cellular environments, i.e not the cytosol) depending on whether they have the right amino acid sequence motifs for these events
What happens if a protein does not fold properly in the ER lumen?
It will fail a quality control check carried out by ER chaperones. Misfolded proteins are not usually allowed to proceed through the secretory pathway
Instead, misfolded proteins are ejected from the ER in a process called retro-translocation (dislocation), and are then degraded in the cytosol
In the Hsp70 family , what are the three chaperones involved with maintaining client solubility?
Hsc70 and Hsp 70 in the cytosol
BiP (GRP78) - binding protein (glucose regulating protein of 78 kDA) in the ER