Membrane contact sites Flashcards
What is the function of the rough ER?
Protein synthesis
What is the function of the smooth ER?
Site of lipid synthesis and calcium storage
What do electron dense areas suggest in electron microscopy?
Lots of protein in the area
What is CLEM?
Correlative light and electron microscopy
How does CLEM work? (4)
- Proteins are labelled
- Look using a microscope and mark where the protein is
- Rapidly freeze and fix the cell for electron microscopy
- Look for the marked protein and visualise the ultrastructure using EM
What techniques have been used to visualise ER membrane contact sites? (3)
- CLEM
- Live cell imaging
- Electron microscopy
What is the structure of ER contact sites? (4)
- Ribosomes are excluded (smooth ER)
- Membranes very close together
- ER contacts can be short or long-lived
- Membranes are connected by long multi-domain proteins which stretch out and hold the membranes together
What is the molecular machinery of ER membrane contact sites?
Tether proteins
What are the features of tether proteins? (4)
- Can be protein-protein
- Can be protein-lipid
- Distance usually 30nm
- Inhibit fusion
How are membrane contact sites defined? (4)
- Absence of fusion
- Must fulfil a function
- Have a defined protein and lipid composition
- Raft-like, enriched in sterols (local rigidity)
What is osbp? (2)
- Tether and lipid transfer protein
- Transfers cholesterol into the ER
What are the functions of ER membrane contact sites? (4)
Provide platforms for:
- Calcium mobilisation
- Lipid transfer
- Signalling
- Organelle division
How do membrane contact sites facilitate calcium mobilisation? (5)
- T-tubule is closely associated with sarcoplasmic reticulum in skeletal and cardiac muscle
- Stim1 is an ER transmembrane protein which is monomeric at high ER Ca2+
- Low ER Ca2+ levels cause oligomerisation of stim1 which interacts with orai1 channel on the plasma membrane
- Orai1 channel is in the membrane where there are high pip2 levels
- Allows transportation of Ca2+ into the ER to replenish ER Ca2+ levels
What is the calcium sensor in the ER?
Stim1
How are membrane contact sites important for lipid transfer? (4)
- Lipids are synthesised in the ER and lipids are delivered to the ER in order to be trafficked to the plasma membrane
- Membrane contact sites allow for non-vesicular transfer of lipids
- Lipid transfer is unidirectional
- Lipid transfer proteins (LTPs) have hydrophobic grooves and may use lipid/ion concentration gradients to promote lipid transfer
What is an example of a disease caused by disruption to lipid transfer? (4)
- Niemann Pick Disease C
- Mutation in a contact-site transmembrane protein which is important for transport of cholesterol from the lysosome into the ER
- Causes accumulation of lipids in the spleen, liver, lungs, bone marrow and brain
- Sphingomyelin accumulates in lysosomes
How are membrane contact sites involved in organelle fission? (2)
- ER surrounds a fission enzyme on mitochondria and contributes to mitochondrial fission
- Mediated by membrane contact sites
What diseases are linked to membrane contact sites? (3)
- TDP43 regulates ER-mitochondria contact sites and is linked to ALS
- REEP1 regulates ER-mitochondria contact sites and is linked to hereditary spastic paraplegia
- Presenilins are involved in Ca2+ exchange between the ER and mitochondria and are mutated in Alzheimer’s
How are membrane contact sites involved in signalling? (3)
- EGFR signalling is attenuated by dephosphorylation and downregulation of the receptors
- EGFR on the endosome is dephosphorylated by a protein tyrosine kinase on the ER membrane which causes EGFR to be incorporated into multi-vesicular bodies and degraded
- Facilitated by contact sites between the ER and endosome
