Unit 2 - Protein Trafficking and Cellular Quality

Unfolded Protein Response (UPR)

Stress sensing and signalling network that maintains homeostasis by rebalancing ER’s capacity

3 UPR Switches

PEAK, ATF6, IRE1

Grp78/BiP

ER stress monitor bound to the 3 switches and covers hydrophobic region during translation

Stress Activation of Grp78/BiP

BiP will leave the switches to bind to unfolded proteins, activating the switches

Activation of IRE1

1. Dimerization and autophosphorylation causes spilicing of XBP1 & RIDD

2. XBP1 can enter nucleus to trascribe CHOP and ER chaperones

IRE1

Localizes inostiol receptors on mitochondrial-ER sites and changes cytoskeletal remodeling via FLNA

Activation of PEAK

1. Dimerization and autophosphorylation causes phosphorylation of eIF2a and increases ATF4 translation

2. eIF2a slows down translation

PERK

Stimulates mitochondria elongation, fusion, and assembly of respiratory chain supercomplexes

PERK ERO1A

Promotes ER-mitochondria contact sites

Activation of ATF6

1. ATF6 exported to golgi via COP 2 vesicles

2. S1P & S2P proteases free transcription factor portion of ATF6 (ATF6N)

Unicellular Eukaryotes Version of UPC

IRE1 and Hac1

Challenges of establish the importance of BiP with IRE1

• Removing BiP binding sites doesn’t affect much

• IRE1 also senses unfolded peptides

• Similarities btwn IRE1 and PERK suggesting a direction mechanism

Lipid Bilayer Stress

Changes in membrane thickness and fluidity activates IRE1 and PERK, ATF6 detects specific sphingolipids

Preemptive UPR

Translation/transcription decreased by IRE1 and PERK via RIDD

Adaptive UPR

IRE1, PERK and ATF4 → XBP1S, ATF4N, ATF6N induce chaperons, foldases, expand ER

Terminal UPR

PERK takes over and ATF4 expresses CHOP causing apoptosis and increase of DR5

Nematodes Neuronal XBP1 Signalling

Activates UPR in distal tissues via synaptic release

Mice Hypothalamus XBP1 from Pro-opiomelanocortin Neurons activates

Liver UPR to restore bioenergetic homeostasis

C. elegans glial XBP1 causes

Nonautonomous metabolic reprogramming

Bioactive Lipids

Messengers to signal a cell-nonautonomous UPR in skeletal muscle

IRE1 Deletion

Increase age associated cognitive decline

IRE1 Present

Promotes neurodegeneration in Huntington’s and Parkinson’s

Transgenic IRE1 Expression

Protects against Alzheimer’s disease

PERK Deletion in Dopamine Neurons

Causes motor and cognitive impairment

Drug Inhibition of PERK

Improves neurodegeneration in Parkinson, Huntington and dementia

Knockout ATF6

Increase loss of dopamine neurons in Parkinson models

Indirect Activation of ATF6

Protects striatal neurons in Huntington’s

Loss of TP53

Stabilizes IRE1

KRAS

Stabilizes IRE1 in pancretic cancer

MYC

Upregulates IRE1 and forms transcriptionally active complex with XBP1S

HIFa

Central hypoxia response mediator

IRE1 promotes tumour angiogenesis by

Increase VEGF-A, IL-6, IL-8 and angiogenin

VEGF-A

Recruits blood vessels to deliver O₂ to centre of tumour

IRE1 Disruption in Cancer

Stops resources to tumours, and overcomes resistance to KBAS-G12C inhibition

Blocking IRE1-XBP1

Stops post chemo tumour regrowth in triple negative breast cancer

Regulated Secretion

Needs stimulus to release proteins

Constitutive Secretion

Protein constantly released

COPII Vesicles

Made of Sec13,16,23,24,31 and moves anterograde from ER to golgi

Sar1

GTPase that initiates formation of COPII coated vesicles at ER membrane

COPI Vesicles

Moves cis golgi to ER

KDEL

4 amino acid sequence on C terminal that indicates it’s a ER resident protein, and lets it be returned via COPi vesicle

ARF

GTPase for COPI and clathrin proteins

Brefeldin A

Inhibits ARF-GDP binding to Sec7 GEF

Clathrin

Moves trans-golgi and plasma membrane vesicles to endosomes

Clathrin + AP1 Complex

Golgi to endosome

Clathrin + AP2 Complex

Plasma membrane to endosome

AP3 Complex

Sorts vesicles into lysosomes

Mannose-6-Phosphate

On soluble lysosomal enzymes after processing in cis-golgi that move them to lysosomes via AP3

Di-Acidic Sequence

On cargo membrane proteins in ER

NPxY Sequence

Recognized by AP2 for endocytosis

Fast Recycling

Direct way of returning membrane component from early endosomes back to plasma membrane

Slow Recycling

Done in the endocytic recycling compartment that takes apart entire protein before retrning back to plasma membrane

Late Endosomes

Moves cargo to lysosomes for final degradation

Endolysosome

Hybrid cellular organelle formed by the fusion of a late endosome and a lysosome

Standard COPII Vesicle Size

60-100nm

COPII Assembly

1. Nucleotide Exchange

2. Inner Coat Formation

3. Outer Coat Assembly

4. Membrane Deformation

Nucleotide Exchange

Sec12 catalyzes GTP binding to Sar1, causing insertion of its helix

Inner Coat Formation

Sar1 GTP recruits Sec23/24, forming inner membrane layer

Sec23-24

Sorts proteins

Outer Coat Assembly

Sec13/31 tetramers form outer layer around budding vesicle

Membrane Deformation

Coat assembly causes bending and budding while activating GTP hydrolysis

Sar1A

Dominant form in tissues

Sar1B

Less common but increase in plasma cells, liver cells, spermatids involved in bigger cargo

Sar1B Mutation

Stops chylomicrons from being secreted

Cylomicrons

Moves fats and cholesterol from small intestine to the body

Tunnel Model

Creates a bridge between ER-ERGIC to move big cargo without vesicle formations

Collar Model

COPII forms rings at ERES base, concentrating cargo into tubular extensions than a vesicle

Tubuler Assemblies

Extended inner coat polymerization, creates straight tubules with fishnet like outer coat

Spherical Vesicles

Cargo interrupts inner coat lattices, causing sphere shape

Main Factor of Membrane Shape

Inner coat polymerization

B Site

Universal, recognizes motifs on all Sec21 paralogues with some preference

C Site

Recognizes conformational epitope on Sec22 to ensure vesicle has fusion machinery

IxM Site

Binds IxM on syntaxin-5, requiring open conformation for SNARE complex

DD Site

Binds to positively charged and hydrophobic amino pairs with paralogue specific preferences

Quality Control in ER Exports

• Misfolded proteins are too big to leave

• Cargos select proteins

• Post-translation changes

• Retrieves ER residents

TANGO1

Helps move large cargo along with COPII proteins

MOTH

MIa, Otoraplin, TALI, TANGO1 important ofr collagen movement

MOTH Functions

• Cargo adaptors

• Diffusion barrier

• Tethering

• Coat Stabilization

Cargo Adaptors

Gets procollagen as C-terminal regions bind Sec23

Diffusion Barrier

Makes rings at ERES to control formation

Tethering

Links ERFIC_53 membranes to ERES for large cargo/direction comms

Coat Stabilization

Competes with Sec31 for Sec23 binding, stops GTP hydrolysis

Cryo EM

Reveals structures

Super-Resolution Fluorescence

Tracks movement

Sec23 Mutation

P382L and M702V causes defective collagen secretion