Lecture 12: ER stress and beyond the ER

Question 1

What are the 5 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 (COPII) vesicles
  
  • 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
  • Bulk flow
    ○ No cargo captured
    ○ Some proteins are retained specifically in the ER
    5 strategies are interdependent that maintain ER homeostasis n reduce homeostasis

Question 2

What is the process of anterograde transport from the endoplasmic reticulum (ER) to the cis-Golgi, and how is it facilitated by COPII-coated vesicles?

Answer 2

• Directionality from ER to cis-Golgi
  
  • Starts w organelle buds called ERES (ER exit site)
    ○ Coated in set of proteins called COP II
  • Transport from the ER to the golgi n further transport
  • COPII organized anterograde transport
    ○ ERES to swell n gets nicked off
    ○ COP II coats captures cytosolic parts of transmembrane receptors
    ○ Receptors for soluble material that pierce the membrane n are captured by COP II coat
    ○ There are also receptors for transmembrane proteins
  • ERES buds off n starts to move towards the Golgi
    ○ Otw these vesicles that bud off start to fuse w the COP II cat-> formation of a structure known as ERGIC (ER-Golgi Intermediate Compartment)
  • When COP II vesicles are close to the cis-Golgi memrane, they shed their coats
    ○ COP II components are recycled
  • ERGIC vesicles fuse w the cis-Golgi -> change in pH -> let receptors to let go of their cargo
  • Result: cargo now delivered by the COPII system into the golgi

Question 3

What is bulk flow in protein transport? Provide advantages of utilizing receptor-independent transport pathways in biotechnology.

Answer 3

• Bulk flow is the transport of proteins into the system in an anterograde manner but doesn’t require any receptors
  
  • Proteins that enter ERES site in receptor independent manner, budded off n transported in forward flow of membrane n vesicles
  • Biotechnological advantage
    ○ Lack of species-specific trafficking signals
    ○ If you direct into ER it will get to the outside of the cell in a receptor-less manner
    ○ Utilizing both flow in different species -> saves effort of working out what receptors normally look like
  • What happens in biotechnology when you transfer a yeast expression?
  • Retention
    ○ Proteins that are excluded from the ER’s exit site -> become retained in the ER

Question 4

What is the retrieval pathway in protein transport?

Answer 4

• Any of the cargo receptors that traffic this pathway to the cis-Golgi need to be returned to the ER to be reused -> retrieval pathway
  
  • Retriever pathway from the cis-golgi back to the ER
  • Can also retrieve proteins incl receptors from the ERGI compounds
  • Protein coat around vesicles that governs this (COPI)
  • COP I coated around vesicles, catches any receptors required for retrieval
    ○ Also recycled
  • ERGIC compounds are a mixture of COPI n COPII vesicles, which allows a nuanced distinction of destinations
  • COP I coats n retrieval
    ○ Captures all the receptors of that were used for the cargo capture step
    ○ Receptors are then brought backwards in retrograde manner -> delivered to ER -> change in pH n dissociated of COP1 allow to be ready for next round of transport

Question 5

How does the cell determine which proteins to retrieve during the retrieval pathway?

Answer 5

• Membrane protein will hv dilysine motif (KK) or close variant
  
  • Other proteins will end up in the wrong place
  • OST has a modified dilysine motif (EKEKSD)
• Cargo receptors will typically hv a KK motif
• What about soluble proteins?
  
  • C terminus of soluble proteins typically end in KDEL motif
  • Proteins that are required for protein folding are retrieved by the golgi

Question 6

How does the RAB6 pathway function as a lipid-handling retrieval system?

Answer 6

• If sending of vesicles in anterograde fashion ->depletnig ER of membrane lipdis
  
  • Extra transport stem that is governed by small G proteins (RAB6)
  • RAB6 organized return by long tubular elements of membrane proteins that hv to be transferred forward n returned to their origin
  • EXAMPLE: Shiga toxin that binds to cell surface n trafficks in retrograde manner to ER utilizes the Rab6 pathway
  • Lipids from the surface of the cell can be retrieved in this pathway, all the way back to the ER
    RAB6 pathway is a lipid-handling retrieval system

Question 7

Provide examples of 3 abiotic stresses

Answer 7

• Heat stress
  
  • Osmotic stress
  • High light intensity

Question 8

Provide examples of 3 biotic stresses

Answer 8

• Infection
  
  • Stress related hormones
    ○ Salicylic acid secreted by a competitor plant, abscisic acid

Question 9

What is the unfolded protein response (UPR)?

Answer 9

• Unfolded protein response: the protein folding capacity of the ER is tightly regulated by a network of signaling pathways
  
  • UPR sensors monitor the ER folding status of proteins in the ER
  • Once UPR is triggered, many physiological events that reduce the stress -> reduce ER folding capacity according to the need

Question 10

Name 3 stressors and where they are found

Answer 10

• Ire1: universal in all known eukaryotes
  
  • PERK: animals n fungi
  • ATF6: animals n fungi
    bZIP28, bZIP17 in plants

Question 11

What is the universal stress sensor in eukaryotes? Explain how it responds to stress

Answer 11

• IRE1 is a universal stress sensor in eukaryotes
  
  • Ire1 is a transmembrane protein
  • ER lumen domain is captured by BiP
  • BiP maintains Ire1 in soluble state but also keeps it inactive
  • Start to increase ER stress -> increase in number of proteins that misfold/unfold
  • BiP detecst this increase n leaves Ire1 -> Ire1 dimerizes n binds to unfolded proteins
  • Increasing ER stress -> increasing Ire1 oligomerization
  • Bringing together Ire1 molecules allows them to activate each other via autotrans phosphorylation
  • Cytosolic domains activate each other
  • Stress sensor multimerizes (increases solubility)
  • Cytosolic activity activates each other

Question 12

How does IRE1 respond to ER stress and what are the downstream effects of its activation?

Answer 12

• Release of BiP by competition of unfolded proteins that are stimulated by stress
  
  • Activation of Ire1
    ○ Not only kinase but also an RNAase (recognizes highly conserved, unusual cytosolic RNA) that has 2 stem loop structures
  • Triangles = sites where Ire1 cuts
  • Ire1 cuts unusual RNA in association w specific ligase
  • 2 exons shown in green are spliced together -> cytosolic intron
  • Splicing 2 exons together creates ORF that is translated to create a transcription factor, which is transported to nucleus
    ○ RESULT: turns on response pathways
    § Upregulate ERAD (now misfolded proteins in the air can be shipped rather more efficiently into the cytosol for destruction)
    § Upregulates folding rates [increasing chaperones in ER]
    § Increases trafficking rate [get rid of unfolded/misfolded proteins]

Question 13

Why is the Ire1 splicing mechanism unusual?

Answer 13

• Ire1
  ○ RNAse activity that recognizes specific cytosolic RNA -> removes highly conserved intron
  ○ Exons are fused using an RNA ligase activity
  
  • Other eukaryotic splicing
    ○ Requires 2 transesterifications coordinated by snRPs n occurs in the nucleus

Question 14

How do we measure ER stress?

Answer 14

• Can stimulate stress in the cell experimentally
  
  • ER protein that is oxidized n reduced DTT
  • DTT
    ○ Breaks disulfide bonds [reducing agent]
    ○ End product: reduced protein n oxidized DTT
    ○ Reducing ER protein -> removing tertiary stabilizing elements -> destabilize the protein -> more likely to unfold
    ○ Therefore DTT stimulates ER stress
  • TG (thapsigargin) Blocks SERCA pumps (sarco/endoplsamic reticulum Ca ATPase): responsible for pumping Ca ions into the ER
    § ER is a Ca store
    ○ If you reduce calcium flow into the ER n maintain calcium flow out of the ER into the cytosol -> start to deplete the ER of Ca
    ○ If you reduce Ca flow into the ER n maintain Ca flow out of the ER into the cytosol Ca dependent chaperones no longer function -> raise the conc of unfolded proteins in the ER
    ○ Depletion of Ca stores -> ER stress
  • Heat shock
    ○ Destabilizes/unfolds some proteins
    ○ Heat shock stimulates ER stress

Question 15

What are the mechanisms by which ER stress is sensed and responded to in both yeast and mammals?

Answer 15

• Stressor is not specific to the organism
  YEAST
  
  • HAC1 unspliced mRNA = Ire1 messenger RNA but for yeast
  • When DTT is added, stress is stimulated
    ○ Cleavage, intron removal to get activated mRNA that can be translated into transcription factor n stimulate reduction of ER stress
    HAC1p protein which is only produced under stress conditions
    MAMMALS
  • TG leads to ER stress
  • XBP1s protein is expressed from spliced XPB1 mRNA

Question 16

Describe the eukaryotic cell’s stress responses when the incubation temperature is raised to 42 degrees

Answer 16

• Heat shock stimulates ER stress
  ○ bZIP60 protein is expressed from spliced bZIP60 mRNA
  
  • Responses
    ○ Increased ERAD (ER-associated protein degradation)
    ○ Increased chaperone production (improved protein folding)
    ○ Increased protein trafficking rates (clearing misfolded proteins from the ER, particularly in yeast)
    RESULT: restoration of ER homeostasis

Question 17

What are the roles of Ire1 in regulating cellular stress and influencing cell fate under severe stress conditions?

Answer 17

• Trigger apoptosis by the junk gene kinase pathway
  
  • Ire1 has general RNase activity (RIDD: regulated IRE1 dependent decay of mRNA)
  • RIDD is regulated Ire1 n decay of mRNA
  • Any mRNA that are close to the ER membrane is mRNA that has been used to translate protein into the ER, degraded non-specifically
  • Cytosolic protein production continues -> less import into the ER -> relieves ER stress
  • Sometimes stress is too great n if Ire1 responses do not control said stress -> triggers apoptosis via JNK signaling pathway
  • Ire1 is a master regulator of cellular stress

Question 18

Compare and contrast PERK n IRE1

Answer 18

• Similarities
  ○ ER domains of PERK n IRE1 share sequence n structural similarity (from yeast n mammals)
  ○ Cytosolic portion of PERK n IRE1 both possess kinase domains that auto phosphorylate in trans
  
  • Differences
    ○ IRE1 activation leads to specific splicing n production of trans activators of transcription
    ○ PERK activation leads to interference w translation
  • If you stop global translation of protein, you reduce stress almost instantly

Question 19

What is the mechanism by which PERK regulates protein synthesis in response to cellular stress?

Answer 19

• Unfolded response caused by stress
  
  • PERK dimerizes n ultimately multimerizes
  • Auto trans phosphorylation that activates PERK
  • Phosphorylation of eukaryotic initiation factor (eIF2α)
    ○ This IF is required for translation of almost all proteins
    ○ When phosphorylated becomes inactivated -> can no longer get the elongation of protein function
    ○ RESULT: protein production globally stops -immediately reduces stress of making more n more proteins

Question 20

How does PERK inhibit global protein translation while promoting the production of specific transcription factors for apoptosis?

Answer 20

• Inhibition of global translation but the stimulation of specific translation
  
  • ATF4 (activating transcription factor 4)
    ○ Transported to the nucleus
    ○ Long chain of events -> expression n activation of CHOP (upstream of apoptosis)
  • PERK can stop protein translation globally but specifically allow production of TF that allow apoptosis

Question 21

How does ATF6 respond to ER stress and contribute to reducing ER stress levels?

Answer 21

• ATF6
  
  • No ER stress
    ○ BiP is bound to ATF6 -> inactivated
    ○ BiP is competed away by unfolded protein
    ○ ATF6 is maintained in the ER
    § When BiP released -> ATF6 transported to Golgi
  • In the Golgi there is a protease that recognized ATF6 n nibbles off the cytosolic domain
  • ATF6 is a TF that goes to the nucleus and upregulated ERAD
    ○ Starts cascade that reduces ER stress

Question 22

Give a brief summary of all the stress pathways found in eukaryotic cells

Answer 22

• PERK
  ○ Reduces translation overall
  ○ Specifies translation of some transcription factors whose roles are stimulate apoptosis
  ○ Oxidative stress pathway downstream of PERK
  ○ PERK can respond to specific stressors other’s cant
  § RESULT: reduces oxidative stress
  
  • Ire1
    ○ Unusual splicing to make TF that upregulates ERAD, folding n protein trafficking into the cell
    ○ Degrades non-specifically
    ○ RNase near the membrane reduces import of proteins into the ER
    ○ Signaling pathway thru JNK that sets off apoptosis
  • ATF6
    ○ Transported to the Golgi
    ○ Upregulates ERAD folding