Lecture 12. The ER as a Portal and Stress Sensor

Question 1

What are the five major interdependent strategies of ER selective transport?

Answer 1

Cargo capture: receptor-mediated export of proteins from the ER to the Golgi complex in coatamer protein II (COPII) vesicles
Bulk Flow: some proteins and lipids are included in COPII vesicles by default
Retention: Prevents proteins from entering the transport vesicles
Retrieval: retrograde transport from the ER-Golgi intermediate compartment (ERGIC)/early Golgi back to the ER
ERAD: Cytosolic elimination of ER proteins that fail quality control

Question 2

What happens with cargo capture and anterograde transport?

Answer 2

1. In the ER, secretory cargo is loaded into COPII (coatamer protein II) transport vesicles at ER exit sites (ERES). This requires export signals in fully folded client proteins and cargo receptor proteins in the vesicle membrane
2. COPII vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC). When COPII vesicles are close to the cis-Golgi membrane, they shed their coats - COPII components are recycled
3. The receptors usually return to the ER by retrieval pathways

Question 3

What happens with bulk flow and retention?

Answer 3

1. Export by bulk flow does not require receptors or export signals. Some soluble and membrane proteins (and membrane lipids) enter COPII vesicles by default
2. There is a biotechnological benefit: foreign proteins directed into the ER are often secreted into the growth medium as soluble proteins that are relatively easy to purify (because they lack species-specific recognition signals)
3. Retention: some proteins are selectively excluded from COPII vesicles

Question 4

What happens in the COPI retrieval pathway?

Answer 4

COPI coated vesicles retrieve transport machinery, cargo receptors, lipid membrane, and escaped ER-resident proteins from ERGIC and the cis-Golgi and delivers it back to the ER

Question 5

What do the retrieved proteins and soluble proteins from COPI coated vesicles have?

Answer 5

Retrieved membrane proteins typically possess a C-terminal dilysine motif (KKXX) or a close variant (e.g OST (human) …EKEKSD)
Retrieved soluble proteins typically have a C-terminal ‘LDEL’ motif (HDEL in yeast and K/HDEL in plants)

Question 6

How is Rab6 involved in retrieval and retrograde transport?

Answer 6

Rab6 organises the return via long tubular elements of membrane proteins transferred forward
Not currently well-characterised (used by bacterial Shiga toxin that binds as a lipid receptor)

Question 7

What are the two categories of stresses that increase misfolding/stimulate unfolding?

Answer 7

Abiotic stresses and biotic stresses

Question 8

What are examples of abiotic stresses?

Answer 8

Heat stress (in plants, fungi and poikilothermic animals especially, but also in
homeothermic animals)
Osmotic stress (Plants: drought, high salinity – can severely impact crop plants)
High light intensity (plants)

Question 9

What are examples of biotic stresses?

Answer 9

Infection (plants, animals, fungi)
Stress related hormones (salicylic acid secreted by a competitor plant, abscisic acid)

Question 10

How can the ER act as a stress sensor in the unfolded protein response (UPR)?

Answer 10

The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated
by a network of signalling pathways - the unfolded protein response (UPR)
UPR sensors monitor the ER folding status of proteins in the ER
Following sensing, the UPR adjusts the folding capacity of the ER according to need

Question 11

What are the multiple sensing mechanisms mediated by/what are three stress sensors?

Answer 11

Ire1 (Inositol-requiring Enzyme 1) – animals, plants, fungi
PERK (PRKR-like endoplasmic reticulum kinase) – animals and fungi
ATF6 (activating transcription factor 6) – animals and fungi (bZIP28 and bZIP17 – plants)

Question 12

What is the structure of Ire1 and what causes Ire1 to dimerise/multimerise?

Answer 12

Ire1 is a transmembrane protein and is bound to BiP in the ER lumen that keeps it inactive
As ER stress occurs and more unfolded/misfolded clients emerge, BiP unbinds from Ire1 stress sensor to bind to the the clients
Ire1 dimerises (and also binds to unfolded proteins)

Question 13

How is Ire1 activated?

Answer 13

Freeing Ire1 from the inactivating effects of BiP allows Ire1 molecules to activate each other through auto-transphosphorylation, cytosolic domains activate each other by kinase activity
Level of stress correlated with level of Ire1

Question 14

How does activated Ire1 splice a specific RNA Ire1 not just a kinase but also an RNase)?

Answer 14

Recognises very specific, highly conserved cytosolic RNA that has two stem loops
Ire1 cuts this RNA with association from a specific ligase, resulting in the two exons being spliced together, leaving an unusual cytosolic intron
The splicing of the two exons creates an open reading frame that’s translated to make a transcription factor which is transported to the nucleus to reduce ER stress through activation of multiple pathways (ERAD, folding and trafficking pathways all upregulated)

Question 15

What is the difference between splicing done by the spliceosome and Ire1?

Answer 15

Most eukaryotic splicing requires two transesterifications coordinated by snRPs, and occurs in the nucleus. Ire1 has RNAse activity that recognises a specific cytosolic RNA, and which removes a highly conserved intron

Question 16

How can ER stress be simulated?

Answer 16

DTT (dithiothreitol) is a reducing agent: it breaks disulphide bonds. If cells are treated with DTT, their ER proteins become destabilised, thus DTT stimulates ER stress
TG (thapsigargin) blocks sarco/endoplasmic reticulum Ca ATPase (SERCA) pumps. Depletion fo ER calcium stores leads to ER stress
Heat shock destabilised/unfolds some proteins and stimulates ER stress

Question 17

How do we know Ire1 has an effect on ER stress?

Answer 17

Ire1 splices HAC1, XBP1 and bZIP60 mRNA
HAC1p (yeast), XBP1s (mammals) and bZIP60 (plant) proteins are all transactivators: once expressed from spliced mRNA, they enter the nucleus and stimulate expression of UPR genes
Restoration of ER homeostasis

Question 18

What is the general RNase activity of Ire1?

Answer 18

RIDD (regulated Ire1 dependent decay of mRNA)
RIDD reduces ER import and so reduces stress
RIDD degrades mRNA in complex with ER-associated ribosomes

Question 19

How is Ire1 involved with signal stimulation?

Answer 19

Stimulates signalling via c-Jun N-terminal kinase (JNK)
Alarm pathways via JNK (a MAPK) can trigger apoptosis

Question 20

What are the similarities between PERK and Ire1 as stress sensors?

Answer 20

The ER domains of PERK and IRE1 share sequence and structural similarity (from yeast and mammals)
The cytosolic portion of PERK and IRE1 both possess kinase domains that autophosphorylate in trans (transmembrane bound)

Question 21

What are the differences between PERK and Ire1 as stress sensors?

Answer 21

IRE1 activation leads to specific splicing and production of transactivators of transcription. However, PERK activation leads to interference with global translation of proteins

Question 22

What is the likely mechanism of PERK?

Answer 22

When no ER stress - PERK is bound to BiP
When there is ER stress - PERK dimerises and autotransphosphorylates
Activated PERK phosphorylates eIF2α (required for translation of nearly all proteins), when phosphorylated, eIF2α is inactivated so no loner get the elongation step in protein synthesis, protein production globally stops

Question 23

What can the phosphorylation of eIF2α cause (ignoring inhibition of global protein production)?

Answer 23

Activating transcription factor 4 (ATF4) translation is promoted
ATF4 is transported to the nucleus and expresses and activated CHOP which is upstream of apoptosis

Question 24

What is the likely mechanism of ATF6?

Answer 24

When no ER stress - ATF6 is bound to BiP
When ER stress - ATF6 is activated and immediately transported to the Golgi
ATF6 is cleaved by a specific protease, releasing ATF6f (transcription factor) that is transported to the nucleus and upregulates BiP, other ER chaperones and ERAD

Question 25

Whats tress receptor is not in plants?

Answer 25

PERK