ER Translocation Flashcards
Features of the ER - rough ER, Smooth ER, why is the ER easy to isolate from the cell?
Rough ER - closely associates with ribosomes
Smooth ER - site of lipid and steroid synthesis
The ER extends throughout the cell so is dense -this makes it easy to isolate by centrifugation
CENTRIFUGATION of the ER - how does it work? What results will the rough and smooth ER display?
Can homogenise the cell and then separate the fractions of the cell based on density
ROUGH ER- more dense - will float @ higher glucose density
SMOOTH ER - will float @ a lower glucose density
Types of signalling sequence and WHY are they needed?
Linear - one long protein
Patched - 3D structure folded protein
Needed to mediate the sorting of proteins
KDEL and KKXX - what are they and where are they found?
KDEL - retrieval signal, linear, found on ER proteins
KKXX - retrieval signal, resident to ER membrane
IDENTIFICATION OF SIGNAL SEQUENCES -
Genetic approach
YEAST - histidine is needed to grow yeast.
Produce mutant line where HISTIDINOL DEHYDROGENASE is targeted to the ER specifically - mutants will still grow in this medium because their translocation machinery will be mutated
IDENTIFICATION OF SIGNAL SEQUENCES - Labelling approach
Label the protein and see if it associated with PARTICULAR ORGANELLES - when a protein has reached its destination its signalling sequence is cleaved off - can separate signalling sequences by running along a glucose gradient
What happens to proteins once they have reached the correct destination?
Signal sequence is cleaved off
SIGNAL SEQUENCE - overall nature ?
Highly variable, 8 NON POLAR amino acids @ the centre of the signalling protein
What is the SRP?
Signal recognition particle - binds to the SIGNAL SEQUENCE which is attached to the protein.
Consists of 6 proteins and 1 RNA.
Where is the protein synthesised and what does it have that allows it to be translocated?
Produced in the ribosome, has SIGNAL SEQUENCE that allows the protein to be translocated to the right place
What is Sec61 and what is its role?
Structural features?
Facilitates the movement of a new protein along the ER.
Pore-like donut shape. PLUGGED in the ABSENCE of RIBOSOMES. The plug is removed when a protein is fed through the pore.
What can be found in the ER environment and why are they needed?
Chaperones - to facilitate folding
Glycosylating enzymes - variety of reasons for increasing complexity of proteins etc etc
This allows CORRECT ASSEMBLY of vesicles and QUALITY CONTROL
What is the importance of quality control?
- Reduces Toxicity
- Prevents transport of abherrent proteins
- Ensures PRECURSOR proteins stay MATURE
What happens AFTER translocation and translation have stopped?
Fully translated protein folds up in the ER lumen
Signal sequence is cleaved off once it has been fed through SEC61
DOUBLE PASS TRANSMEMBRANE PROTEINS - what does the signal sequence do in the case of these proteins?
Behaves as an anchor — anchors protein to the cell membrane instead of being cleaved away.
Why must some proteins be degraded?
- Misfolding
- If not correctly folded, UGGT tries again —> if not terminal misfolding is seen and the protein is TAGGED FOR DEGRADATION
What is the main role of chaperone proteins?
EXAMPLES?
Aids in folding and ASSEMBLY into COPII coated VESICLES.
- ERP57
- CALRETICULUM
What is the UPR and why does it happen?
Unfolded Protein Response
-Cell stress response - results in transcriptional activation of genes which produce MORE CHAPERONES and result in MORE ER SYNTHESIS
What is ERAD and what does it do?
PROTEIN which mediates ER-associated protein degradation
Adds UBIQUITIN to a protein which tags it for degradation
uses REVERSE TRANSLOCATION MACHINERY
Examples of sensors from misfolded proteins that become activated to induce UPR
-IRE1
-PERK
-ATF6
These activate in misfolded proteins and cause an INCREASE IN THE FOLDING CAPACITY OF THE ER
ER STRESS prodeath and prosurvival pathways- which becomes stronger with time
The longer the UPR is present the stronger the pro-death signals become which induce apoptosis in cells
Different trans factors are active in both pathways
ATF6 and Bip - what happens in response to stress?
No stress - AFT6 bound to Bip
Stress - AFT6 dissociates from Bip, Bip is cleaved in the Golgi and this STIMULATES THE PRODUCTION OF CHAPERONES
Cholesterol homeostasis - what happens in the presence of high cholesterol?
SREBP is retained in the ER by SCAP/Insig complex
Insig = chaperone
Cholesterol homeostasis - what happens in the presence of low cholesterol?
SCAP/Insig dissociate – SREBP transits to the Golgi and is cleaved —> enters the cell nucleus and increases transcription of genes which are needed to increase conc of cholesterol
HOW is the AUTOPHAGIC PATH involved?
Why is this important?
Sometimes too much ER produced – this is engulfed by macrophages.
Defects in this system can result in disease
OUTLINE TRANSLOCATION OF PROTEINS ALONG THE ER
1) Protein is synthesised at the ribosome WITH signalling sequence
2) Signal is recognised by SIGNAL RECOGNITION PARTICLE (SRP)
3) SRP and protein form a complex which binds to SRPreceptor in the Rough ER, PAUSING translation
4) Complex is brought close to the translocator, where SRP then dissociates from the SRPreceptor
5) SRP is displaced and translocation/translation continues
