Lecture 12

• ER Function

  The ER serves as the initial checkpoint for proteins destined for the secretory pathway, where proteins must have a signal peptide to be targeted to the ER
• Protein Folding

  Within the ER, protein folding is tightly regulated by a network of chaperones and co-chaperones, ensuring that only properly folded proteins proceed
• Quality Control

  Proteins that fail to fold correctly are marked for destruction by the ubiquitin-proteasome system (UPS) in the cytosol
• Anterograde Trafficking

  1. Forward movement of proteins from the ER to the Golgi apparatus and beyond

  2. Cargo Capture: Involves the export of proteins from the ER to the Golgi complex via coatamer protein II (COPII) vesicles

  3. Bulk Flow: Some proteins and lipids are included in COPII vesicles by default without specific signals

  4. Retention and Retrieval: Certain proteins are selectively retained within the ER or retrieved from the Golgi back to the ER via retrograde transport
• ERAD (ER-associated Degradation)

  Targets and eliminates misfolded ER proteins via the cytosolic UPS
• COPII Vesicles

  Transport proteins from ER exit sites (ERES) to the ER-Golgi intermediate compartment (ERGIC) and eventually to the cis-Golgi, shedding their coats as they fuse and transition towards the Golgi
• COPI and Rab6

  1. COPI vesicles are involved in retrograde transport, retrieving components like cargo receptors and escaped ER-resident proteins back to the ER

  2. Rab6, a small GTPase, regulates another retrograde route, possibly for returning specific lipids to the ER
• Molecular Signatures for Retention and Retrieval

  • Soluble proteins often have a C-terminal 'KDEL' motif
  • Membrane proteins may have a C-terminal dilysine motif
• Directing foreign proteins into the ER can lead to their secretion into the growth medium, making them easier to purify due to the lack of species-specific recognition signals
• Function of the UPR

  The UPR is a signaling network that regulates the protein folding capacity of the ER. It monitors the folding status of proteins within the ER and adjusts its capacity according to cellular needs
• UPR Sensors


  • Ire1 (Inositol-requiring Enzyme 1)
  • PERK (PRKR-like endoplasmic reticulum kinase)
  • ATF6 (activating transcription factor 6)
  • Plant-specific bZIP proteins like bZIP28 and bZIP17
• Ire1 Activation

  1. Under no stress, Ire1 is bound to BiP (binding immunoglobulin protein)

  2. With low ER stress, Ire1 dimerizes and binds unfolded proteins, leading to increased oligomerization with increasing stress

  3. The cytosolic domains of Ire1 transautophosphorylate, activating its RNAse activity
• RNA Splicing by Ire1

  1. Activated Ire1 splices specific RNA sequences, leading to changes in gene expression that help reduce ER stress

  2. In yeast, Ire1p splices HAC1 mRNA; in mammals, it splices XBP1 mRNA; in plants, it splices bZIP60 mRNA
• Methods to Stimulate ER Stress

  • Dithiothreitol (DTT): A reducing agent that breaks disulfide bonds, destabilizing proteins and simulating ER stress
  • Thapsigargin (TG): Blocks sarco/endoplasmic reticulum Ca2+ ATPases (SERCA), depleting ER calcium stores and inducing stress
  • Heat Shock: At temperatures like 42°C, some proteins unfold or destabilize, mimicking conditions of ER stress
• Monitoring ER Stress

  1. Observing the splicing activity of Ire1

  2. DTT and TG Treatments: Influence on the splicing of specific mRNAs like HAC1 in yeast, XBP1 in mammals, and bZIP60 in plants

  3. Increased ER-associated protein degradation (ERAD)

  4. Enhanced production of chaperones to improve protein folding

  5. Increased rates of protein trafficking to clear misfolded proteins from the ER
• RIDD (Regulated IRE1 Dependent Decay)

  Reduces mRNA levels associated with proteins entering the ER, thus decreasing stress
• 1. Jun N-terminal kinase (JNK) Activation

  Ire1 can also stimulate signaling pathways leading to apoptosis, involving MAPK proteins like JNK
• PERK Activation Mechanism

  • Under non-stress conditions, PERK is bound to BiP
  • Upon ER stress, PERK dimerizes and autophosphorylates
  • Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), leading to a global reduction in translation, which helps reduce the overall protein load in the ER, alleviating stress
• ATF6 Activation and Function

  • Under non-stress conditions, ATF6 is bound to BiP
  • When misfolded or unfolded proteins accumulate, ATF6 is released from BiP and moves to the Golgi apparatus
  • In the Golgi, ATF6 is cleaved, releasing ATF6f, a transcription factor that moves to the nucleus to activate genes enhancing protein folding, ERAD (ER-associated degradation), and upregulating ER chaperones
• Integrated Stress Response

  • The pathways mediated by PERK, Ire1, and ATF6 interact and integrate to form a robust response to ER stress
  • Activation of these pathways leads to various outcomes: PERK - Reduction in global protein synthesis, selective translation of ATF4, leading to adaptive responses or apoptosis; Ire1 - Activation leads to splicing of mRNA (like XBP1 in mammals), which enhances the cell's capacity to deal with unfolded proteins through increased ERAD, folding, and trafficking; ATF6 - Activation enhances the cell's folding capacity and ERAD
• Plants do not have PERK; instead, they rely on Ire1 and its interaction with other transcription factors like bZIP28 and bZIP17, which are crucial under conditions such as salt stress