Protein Modification: The ER as a Portal and Stress Sensor

Beyond the ER

  • The ER (Endoplasmic Reticulum) is a central organelle in the cell responsible for protein folding and modification.
  • Folded proteins in the ER can have different fates, including transport to the Golgi apparatus, secretory vesicles, late endosomes, lysosomes, and the plasma membrane.
  • Vesicular transport is a key mechanism for moving proteins between these destinations.

Trafficking

  • Anterograde trafficking (forward): from the ER to other destinations.
  • Retrograde trafficking: from the ER-Golgi intermediate compartment (ERGIC) and early Golgi back to the ER.
  • Gated transport: to and from the nucleus. (LF104)
  • Transmembrane transport: to plastids, peroxisomes, and mitochondria. (LF206 and BS318)

ER Selective Transport: Five Major Interdependent Strategies

  • Cargo capture: Receptor-mediated export of proteins from the ER to the Golgi complex in coatamer protein II (COPII) vesicles (anterograde, vesicular trafficking).
  • Bulk flow: Some proteins and lipids are included in COPII vesicles by default.
  • Retention: Prevents proteins from entering the transport vesicles (some proteins are excluded from further transport).
  • Retrieval: Retrograde transport from the ER-Golgi intermediate compartment (ERGIC) /early Golgi back to the ER (via COP1 vesicles).
  • ERAD: Cytosolic elimination of ER proteins that fail quality control.
  • Reference: Barlowe and Helenius (2016). Cargo Capture and Bulk Flow in the Early Secretory Pathway. Annual Review of Cell and Developmental Biology, 32, 197-222.

Cargo Capture and Anterograde Transport

  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.

Bulk Flow and Retention

  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.

Retrieval and Retrograde Transport (COPI)

  • COPI coated vesicles retrieve transport machinery, cargo receptors, lipid membrane, and escaped ER-resident proteins from ERGIC and the cis-Golgi.
  • Retrieved membrane proteins typically possess a C-terminal dilysine motif (KKXX) or a close variant. E.g., human OST …EKEKSD
  • Retrieved soluble proteins typically have a C-terminal ‘KDEL’ motif (HDEL in yeast, and K/HDEL in plants).
  • Examples:
    • BiP (human) …..DTAEKDEL
    • ERp57 (human) ….. KKKAQEDL
    • CRT (human) ….. PGQAKDEL

Retrieval and Retrograde Transport (Rab6)

  • There is also a retrograde route governed by the small GTPase Rab6 protein: these tubular elements are independent of COPI.
  • Not currently well-characterised.
  • It is used by the bacterial Shiga toxin that binds a lipid receptor: is the Rab6-controlled route’s purpose to return specific lipids to the ER?
  • Proteins not retrieved are sorted for further destinations

Summary of ER Functions

  • Cytosol:
    • Protein folding is regulated by a network of chaperones and co-chaperones.
    • Proteins are destroyed by the UPS (ubiquitin-proteasome system).
  • ER, the gateway to the secretory system:
    • ER targeting requires a signal peptide, which is removed after targeting (after entry into the ER).
    • ER protein folding is regulated by a network of chaperones and co-chaperones.
    • ER proteins are destroyed by the UPS (in the cytosol)- if a protein fails ER quality control, it’s recognised, unfolded, threaded out of the ER into the cytosol and is dealt with by the UPS.
    • Selective transport to further destinations

ER Stress and the Unfolded Protein Response (UPR)

  • What happens if protein unfolding/misfolding is increased by stress?
  • 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.
  • If detects a load of folding greater than expected- signlas to change cellular physiology to reduce

Stresses That Increase Misfolding/Stimulate Unfolding

  • Abiotic stresses:
    • Heat stress unfolds proteins (in plants, fungi and poikilothermic animals especially, but also in endothermic animals)- hard to regulate temperature
    • Osmotic stress unfolds proteins (Plants: drought, high salinity – can severely impact crop plants)
    • High light intensity (plants)- absorption spectrum of chlorophyll has 2 peaks and in between burning of plants occurs
  • Biotic stresses:
    • Infection (plants, animals, fungi) increase temperature as well as other stressors increasing unfolded protein load
    • Stress related hormones (salicylic acid secreted by a competitor plant, abscisic acid)

UPR Sensing Mechanisms

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

Ire1

  • Under low ER stress, BiP is bound to Ire1.
  • Increasing ER stress stimulates increasing Ire1 oligomerisation.
  • The cytosolic domains of Ire1 transautophosphorylate, activating Ire1 RNAse activity.
  • Activated Ire1 splices a specific RNA, leading to the production of a transcription factor.
  • This transcription factor goes to the nucleus to promote reduction of ER stress through ERAD, folding, and trafficking.

Ire1 Splicing Mechanism

  • Ire1 uses an unusual splicing mechanism.
  • Most eukaryotic splicing requires two transesterifications coordinated by snRPs and occurs in the nucleus.
  • By contrast, Ire1 in plants, mammals and fungi has RNAse activity that recognises a specific cytosolic RNA and removes a highly conserved intron.
  • The exons are fused using an RNA ligase activity.

Measuring ER Stress

  • Treatment with DTT destabilises ER proteins. Thus, DTT stimulates ER stress. SS+SHSHS S + SH SH
  • TG (thapsigargin) blocks sarco/endoplasmic reticulum Ca2+Ca^{2+} ATPase (SERCA) pumps.
  • Depletion of ER calcium stores leads to ER stress.
  • Ca++ cytosol ER lumen SERCA
  • Heat shock (e.g., 42°C) destabilises/unfolds some proteins.
  • Heat shock stimulates ER stress.

Monitoring Ire1-Mediated Splicing

  • In yeast, activated Ire1p splices HAC1 mRNA.
  • In mammals, activated Ire1 splices XBP1 mRNA.
  • In plants, activated Ire1 splices 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.

Responses to ER Stress

  • Increased ERAD (ER-associated protein degradation)- lowers load of misfolded proteins
  • Increased chaperone production (improved protein folding)- alters the folding capacity of the ER
  • Increased protein trafficking rates (clearing misfolded proteins from the ER, particularly in yeast)- increased protein trapping rates to clear ER of misfolded proteins
  • Results: restoration of ER homeostasis

Ire1 and RIDD

  • Ire1 also has a general RNase activity (RIDD).
  • Regulated IRE1 dependent decay of mRNA (RIDD).
  • Reduces ER import and so reduces stress.
  • RIDD degrades mRNA in complex with ER-associated ribosomes

Ire1 and JNK

  • Ire1 can also stimulate signalling via c-Jun N-terminal kinase (JNK).
  • Can trigger apoptosis
  • Alarm pathways via JNK (a MAPK) - see later

PERK

  • PERK and IRE1 Share similarities:
    • 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.

PERK - Differences from IRE1

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

PERK Mechanism

  • Activated PERK phosphorylates eIF2α.
  • Activated eIF2α reduces translation globally – and this reduces protein expression and relieves stress.
  • However, some RNAs are preferentially translated, e.g. ATF4
  • ATF4 = activating transcription factor 4
  • CHOP expressed
  • APOPTOSIS

ATF6

  • ATF6 is transported to the Golgi.
  • ATF6 is cleaved.
  • ATF6f is a transcription factor that goes to the nucleus to promote expression of Bip and other ER chaperones, as well as ERAD components.

Summary: Three Interacting Pathways in Mammals

  • PERK leads to phosphorylation of eIF2α, reducing translation and increasing ATF4 expression, which can lead to apoptosis.
  • Ire1 leads to splicing of XBP1, promoting ERAD, folding, and trafficking.
  • ATF6 is cleaved to ATF6f, which promotes ERAD and folding.
  • Cross-talk and integration between these pathways.

Summary: ER Stress Response in Plants

  • Plants have Ire1 and ATF6-like pathways, but no PERK.
  • bZIP28 and bZIP17 are involved in the salt stress response.

Study of ER and Trafficking

  • See Protein Targeting BS318 for more details.