The Endomembrane system II Flashcards
(28 cards)
How is compartmental identity maintained i.e. what is required
Distinct set of enzymes and lipids to each compartment
Efficient sorting of resident and cargo molecules
Accurate targeting of carrier vesicles
Signals on proteins to sort them into the correct transport vesicles or to retain them in correct compartment
Signals on transport vesicles to target them to correct compartments
How are transport vesicles formed
By assembly of protein coats
Coat is formed on cytoplasmic face of membrane
What are the functions of a coat
To deform membrane and package cargo
Several types of vesicles such as COPI, COPII, and Clathrin
Each type of vesicle has different transport function
What do COPII vesicles do
Transfer proteins and lipids from the ER to the Golgi
The COPII proteins assemble into a lattice on the ER
What are ER resident proteins
They are retained in the ER whereas all other proteins are exposed in COPII vesicles for secretion or transport to other organelles. Export from the ER does not require a signal - can occur by default or bulk flow
Introduction of bacterial proteins in the endoplasmic reticulum
Outline how this is done and what it therefore demonstrates
- Bacterial proteins are unlikely encode signals for export from ER
1. Take a bacterial gene, and fuse it onto a DNA sequence encoding a eukaryotic signal peptide for targeting to ER
2. Add eukaryotic transcription signals (promoter, polyA signal) and introduce into eukaryotic cell
Bingo! The bacterial proteins are secreted when introduced into ER.
What does this imply
Implies that no signal is required for export or secretion
Implies that a signal is required for ER residency
What happens then the KDEL sequence has been experimentally removed?
What does this show?
Primary signals are necessary and sufficient to signal ER residency. Where the KDEL sequence has been removed in genetically modified ER resident proteins, the cell secretes these proteins albeit at a much slower rate than normal secretory proteins
Suggests another mechanism independent of the KDEL retrieval pathway that retains these proteins in the ER
The sequence of ER-resident proteins is necessary and sufficient. How can this be shown?
Necessary - Deletion of the signal (as in previous card) results in secretion of the ER protein
Sufficient - transplanting the signal to a secreted protein causes its retention so the signal is sufficient too
How do these signals maintain ER proteins in the ER?
Clue: residency signals are actually retrieval signals
ER resident proteins are not actually prevented from leaving - relies on continuous retrieval from the cis-Golgi
COP-I vesicles selectively bind to the retrieval signals on escaped ER-membrane proteins and return them to ER
ER-retrieval signals are found on cytoplasmic tails of ER-membrane proteins and the COP-I coat proteins bind to retrieval signals on membrane proteins
The KDEL receptor in the cis-Golgi membrane binds to KDEL signal on the lumen side and to COP-I on cytoplasmic side
Describe KDEL receptor recycling
- Cargo binding and release regulated by lumen pH
KDEL receptor binds cargo at slightly acidic pH but releases at neutral pH
KDEL receptor recycling to cis-Golgi in COP-II vesicles
Describe KDEL receptor recycling
- Cargo binding and release regulated by lumen pH
KDEL receptor binds cargo at slightly acidic pH but releases at neutral pH (i.e. affinity for the proteins vary depending on the pH)
KDEL receptor recycling to cis-Golgi in COP-II vesicles
How does TMD signal promote Golgi localisation through protein-lipid interactions
The lipid compositions of the ER Golgi and PM are different –> different membrane thicknesses
PM>Golgi>ER and the TMDs therefore also differ in length
SO
How does TMD signal promote Golgi localisation through protein-lipid interactions
Hint: think about the difference between PM, Golgi, and ER
The lipid compositions of the ER Golgi and PM are different –> different membrane thicknesses
PM>Golgi>ER and the TMDs therefore also differ in length
SO…
The subcellular location depends on the LENGTH of the TMD domain
Please outline the process of receptor-mediated cholesterol endocytosis
- Cholesterol is transported as LDL particles bound by LDL receptor at PM and endocytosed
- An endocytosis signal in the cytoplasmic tail of the LDL receptors binds the membrane-bound adaptor protein AP2
- LDL receptors are proteins made by the cell anticipating cholesterol delivery and they diffuse until they associate with clathrin coated pits.
- LDL particles bound to LDL receptors in the coated pits are internalised in coated vesicles
- Then the vesicles shed their Clathrin coat and deliver contents to early endosomes. Low pH in early endosomes leads to release of LDL
-LDL is delivered by late endosomes to lysosomes
-
What happens to the LDL in the lysosome
Hydrolysed to free cholesterol available for new membrane synthesis
How do the clathrin coated pits select their cargo?
By binding to the cytoplasmic tails of the receptors
What are the two major protein components of CCVs?
Clathrin complex - outer shells
Adaptin complex
inner layer, selects the cargo
What do adaptins do?
Recognise signals in cytoplasmic domains of receptors
Describe FHC
Lack the LDL receptor gene or have mutations that alter the sequence of the internalisation signal in the receptor Therefore cholesterol not taken up from the bloodstream
What is the clathrin triskelion
Each clathrin subunit consist of three large and three small polypeptide chains that together form a three legged structure called a triskelion. The triskelions assemble into a basket like framework to form coated pits on the cytosolic surface of membranes and can self-assemble.
It is a very stable structure that can achieve a high radius of curvature
Clathrin coated vesicles are targeted to the early endosome
Receptors release the cargo and is recycled to the PM
Cargo travels to late endosome and lysosome for degradation
EE has important sorting function
How does the mannose 6-phosphate receptor sort lysosomal hydrolases in the trans Golgi network
Clue: what does the M6P protein receptor bind to?
- Mannose 6-phosphate groups are phosphorylated to the N-linked oligosaccharides of the enzymes as they pass through the lumen of the cis Golgi network.
- Transmembrane M6P receptor proteins recognise the M6P phosphate groups and bind to the lysosomal hydrolyses on the lumenal side of the membrane AND to adaptor proteins (AP1 – *note endocytosis uses adaptin AP2) in assembling clathrin coats on the cytosolic side –> the receptors help to package the enzymes into CCVs that bud from the TGN
- Signals in the cytoplasmic tail of the M6P receptor direct the protein to the LATE endosome (low pH)
How are they distinguished initially by a signal patch in their tertiary structure
The recognition signal is a cluster of neighbouring amino acids – the signal patch
Most hydrolases contain multiple oligosaccharides – therefore acquire many M6P groups providing a high affinity signal for the M6P receptor.
What happens to the M6P receptor after releasing cargo into the late endosome
The receptor is recycled back to the trans Golgi
