ER Translocation

Question 1

Features of the ER - rough ER, Smooth ER, why is the ER easy to isolate from the cell?

Answer 1

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

Question 2

CENTRIFUGATION of the ER - how does it work? What results will the rough and smooth ER display?

Answer 2

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

Question 3

Types of signalling sequence and WHY are they needed?

Answer 3

Linear - one long protein
Patched - 3D structure folded protein
Needed to mediate the sorting of proteins

Question 4

KDEL and KKXX - what are they and where are they found?

Answer 4

KDEL - retrieval signal, linear, found on ER proteins

KKXX - retrieval signal, resident to ER membrane

Question 5

IDENTIFICATION OF SIGNAL SEQUENCES -

Genetic approach

Answer 5

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

Question 6

IDENTIFICATION OF SIGNAL SEQUENCES - Labelling approach

Answer 6

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

Question 7

What happens to proteins once they have reached the correct destination?

Answer 7

Signal sequence is cleaved off

Question 8

SIGNAL SEQUENCE - overall nature ?

Answer 8

Highly variable, 8 NON POLAR amino acids @ the centre of the signalling protein

Question 9

What is the SRP?

Answer 9

Signal recognition particle - binds to the SIGNAL SEQUENCE which is attached to the protein.
Consists of 6 proteins and 1 RNA.

Question 10

Where is the protein synthesised and what does it have that allows it to be translocated?

Answer 10

Produced in the ribosome, has SIGNAL SEQUENCE that allows the protein to be translocated to the right place

Question 11

What is Sec61 and what is its role?

Structural features?

Answer 11

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.

Question 12

What can be found in the ER environment and why are they needed?

Answer 12

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

Question 13

What is the importance of quality control?

Answer 13

• Reduces Toxicity
• Prevents transport of abherrent proteins
• Ensures PRECURSOR proteins stay MATURE

Question 14

What happens AFTER translocation and translation have stopped?

Answer 14

Fully translated protein folds up in the ER lumen

Signal sequence is cleaved off once it has been fed through SEC61

Question 15

DOUBLE PASS TRANSMEMBRANE PROTEINS - what does the signal sequence do in the case of these proteins?

Answer 15

Behaves as an anchor — anchors protein to the cell membrane instead of being cleaved away.

Question 16

Why must some proteins be degraded?

Answer 16

• Misfolding
• If not correctly folded, UGGT tries again —> if not terminal misfolding is seen and the protein is TAGGED FOR DEGRADATION

Question 17

What is the main role of chaperone proteins?

EXAMPLES?

Answer 17

Aids in folding and ASSEMBLY into COPII coated VESICLES.

• ERP57
• CALRETICULUM

Question 18

What is the UPR and why does it happen?

Answer 18

Unfolded Protein Response
-Cell stress response - results in transcriptional activation of genes which produce MORE CHAPERONES and result in MORE ER SYNTHESIS

Question 19

What is ERAD and what does it do?

Answer 19

PROTEIN which mediates ER-associated protein degradation
Adds UBIQUITIN to a protein which tags it for degradation
uses REVERSE TRANSLOCATION MACHINERY

Question 20

Examples of sensors from misfolded proteins that become activated to induce UPR

Answer 20

-IRE1
-PERK
-ATF6
These activate in misfolded proteins and cause an INCREASE IN THE FOLDING CAPACITY OF THE ER

Question 21

ER STRESS prodeath and prosurvival pathways- which becomes stronger with time

Answer 21

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

Question 22

ATF6 and Bip - what happens in response to stress?

Answer 22

No stress - AFT6 bound to Bip

Stress - AFT6 dissociates from Bip, Bip is cleaved in the Golgi and this STIMULATES THE PRODUCTION OF CHAPERONES

Question 23

Cholesterol homeostasis - what happens in the presence of high cholesterol?

Answer 23

SREBP is retained in the ER by SCAP/Insig complex

Insig = chaperone

Question 24

Cholesterol homeostasis - what happens in the presence of low cholesterol?

Answer 24

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

Question 25

HOW is the AUTOPHAGIC PATH involved?

Why is this important?

Answer 25

Sometimes too much ER produced – this is engulfed by macrophages.
Defects in this system can result in disease

Question 26

OUTLINE TRANSLOCATION OF PROTEINS ALONG THE ER

Answer 26

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