Endomembrane II Flashcards
The early endosome is involved in
cargo sorting
The recycling endosome is involved in
recycling vesicles
Transport between the Golgi and the late endosome is
- secretory
- results in endosomal convergence.
The Golgi is involved in
- carbohydrate synthesis
- cargo sorting
The endoplasmic reticulum is involved in
- protein translocation
- lipid synthesis
How is macromolecular transport achieved in the cell?
The discrete secretory pathway and endocytic pathways interact
What are the resident molecules?
characteristic set of functional proteins and lipids of the secretory and endocytic pathways
What is the cargo?
proteins and lipid
What else in necessary for the secretory and endocytic pathways, excluding resident molecules and cargo?
compartments to receive the cargo
What are the consequences of having disparate secretory and endocytic pathways?
cell requires an efficient sorting system of signals to distinguish resident from cargo molecules and maintain compartmental identity.
What do resident/cargo discrimination systems usually reply upon?
signals to fuse vesicles to the appropriate protein
Compartmental specialisation relies upon 4 precedents:
I) resident and cargo molecules are efficiently sorted
ii) carrier vesicles are accurately targeted
iii) proteins are sorted and retained in the correct transport vesicles and compartments by protein signalling
iv) transport vesicles are targeted to the correct compartments using signalling
Transport vesicles are designed to
transport cargo.
Describe transport vesicle formation
- self-assembly of protein coats from coat subunits on the cytoplasmic side of the endomembrane system
- budding of this membrane from the organelle lumen
- disassembly of this coat, as the vesicle fuses to the ER.
Describe the effect of the protein coat
- aids the deformation of the membrane bilayer
- necessary for vesicles with large membrane curvature.
COP-II vesicles basics
- transfer secretory proteins and lipids with signal membranes from the ER lumen or membrane to the cis Golgi
- leave ER resident proteins behind.
Describe COP-II vesicle protein coats
- composed of two layers which coat the membrane I) the adaptor layer and ii) the cage layer
- vary for 50 to >90nm in diameter.
Under biological logic, a dynamic entity must have a minimum of two fates
Describe the COP-II adaptor layer
made of adaptor proteins that interact with the membrane
Describe the COP-II cage layer
- made of rod-like cage proteins that assemble into a lattice
- bending allows curvature that will stabilise the structure
- can visualised under cross-section using TEM or cyro-electron microscopy
Describe COP-II vesicle transport
when sorting, the residents are retained in the ER and become enriched, whereas the cargo are exported for secretion at the plasmamembrane, or transport to other organelles.
Describe an experiment that tested COP-II vesicle transport
- introducing bacterial proteins and artificial peptides into the ER: fusing a bacterial gene onto a eukaryotic signal peptide gene sequence targeting the ER, and adding eukaryotic transcription signals such as promotor and polyA signal, then introducing into a eukaryotic cell
- ER secretion occurred for bacterial proteins
- ER resident proteins have short amino acid sequences signalling residency which are necessary and sufficient
Why is often useful to introduce bacterial proteins in endomembrane experiments?
unlikely to carry genes coding for organelle export functions, as they do not contain organelles
How were the short amino acid sequences signalling residency of resident proteins proven to be necessary?
genetic deletion of the signal resulted in secretion
How were the short amino acid sequences signalling residency of resident proteins proven to be sufficient?
transprotein transplanting of the signal onto a cargo protein results in retention
Export is the
default state, not requiring a signal; occurs by ‘bulk flow’
ER residency is
the signalled state
ER residency is in fact
continuous retrieval from the cis-Golgi in a form of recycling
Describe continuous retrieval of resident proteins from the cis-Golgi
achieved by COP-I coated vesicles, whose coat proteins recognise and bind to ER retrieval signals, returning them to the ER
ER retrieval signals
- short lysine/arginine enriched proteins xRR and KKxx
- on the cytoplasmic tails of escaped ER-membrane proteins
COP-I is
a selective vesicle for secreted cargo membrane proteins – this is cargo selection.
Describe the mechanism of COP-I interaction
- allows interaction of its cytoplasmic head and the cis-Golgi membrane on the cytoplasmic side through the cis-Golgi’s KDEL receptor
- binds to the KDEL ER-retrieval signal on the soluble proteins of the ER lumen on the lumenal side
Describe COP-I
- pre-assembled coatomer recruited to the membrane, forming a curved triad subunit
- triads are connected by flexibly attached domains that allow self-assembly, and propagation of membrane bending
- visualised under cryo-electron tomography
Coatomer
heteroheptameric complex
Describe COP-I membrane
KKxx cargo-motif binding sites
Cargo binding and release is regulated by
- lumenal pH
- KDEL-receptor binds to the cargo for recycling at pH 6-6.5 but releases at 7. – COP-I vesicles can recycle KDEL-receptors to the cis-Golgi from the ER.
pH of the cis-Golgi
6-6.5
pH of the ER
7
Resident membrane proteins of the Golgi are
- made in the ER
- transported as cargo to the Golgi, but not to the plasmamembrane
– signalled fate
Describe Golgi residence
resident Golgi enzymes contain a very distinctive large catalytic lumen domain for their enzymatic function of polysachharide synthesis and modification
Describe Golgi resident localisation signals
single TMDs in the membrane, and the short N-terminal cytosolic tail domain
TMD subcellular localisation is promoted via
- promoted via protein–lipid interactions
- dependent upon the different lengths of the ER , Golgi and plasmamembrane TMDs.
ER TMD length
approximately 15-17aas
Golgi TMD length
approximately 17-20aa
Plasmamembrane TMD length
> 20aas
TMD has been found to be both … and … for Golgi localisation.
necessary … sufficient
What happens if TMDs are longer?
localised to the plasmembrane
What happens if TMDs are shorter?
localised to the ER
If ER TMDs are made longer, or plasmamembrane TMDs are made shorter…
they are localised to the Golgi
Describe cholesterol transport
- small lipid droplets with phospholipids and protein in hydrophilic environments
- endocytosed as LDL particles
- bound by LDL receptor proteins with LDL binding sites on the plasmamembrane
- LDL receptor is collected at the plasmembrane into pits coated in clathrin and other coat-associated proteins on the cytosolic side
- the coated pit can be actively pinched off into a CCV, where its clathrin coat dissociates with the aid of a large protein suite
Where is cholesterol transported?
Blood vessels
LDL
- low density lipoprotein
- cholesteryl ester molecules surrounded by cholesterol and phospholipid molecules
- surface protrusion and specific head composition
- 22nm
Describe Familial hypercholesterolaemia
- lack the gene to synthesise this LDL receptor proteins, or exhibit mutations that alter the cytoplasmic domain of the LDR receptor
- prevents interaction with adaptin
- cholesterol cannot be internalised from the bloodstream
- cholesterol build up
- early heart attack
CCV
Clathrin Coated Vesicle
Describe CCVs
two proteinous layers: the inner, AP2 complex, and outer clathrin complex
AP2
- adaptin2
- selective
- binds to the cytoplasmic tail domains of cargo receptor complices
- recruits outer clathrin complex
Outer Clathrin complex
- bends the membrane
- provides structure
Describe adaptin binding
- by clathrin triskelions
- creates a high radius of curvature that is extremely stable, and requires a lot of proteins to break
Describe clathrin triskelions
- molecular assembly of six differently-weighted protein subunits
- spontaneously self-assembling into small, closed clathrin lattices
- can be viewed under cryo- and deep-etch electron microscopy
- always present in the cell
Describe what happens to CCVs post-endocytosis and coat removal
- targeted to and merge with the early endosome for sorting
- receptor-cargo complex dissociates
- LDL receptor is repackaged and recycled, via the recycling endosome, back to the plasmamembrane
- cargo travels to the late endosome and lysosome for degradation by the lysosomal hydrolases
- endocytic cargo and the hydrolases are trafficked to the late endosome
- remaining cargo is trafficked to the lysosome
What causes receptor-cargo dissociation?
acidic pH relative to the plasmamembrane and extracellular space
Give a lysosome hydrolase
Cathepsin-D
Describe the structure of Cathepsin-D
- inactive precursor proenzyme form is synthesised in the ER with a distinguishing signal patch of a specific mannose residue on the N-glycan in the tertiary structure
Describe the action of Cathepsin-D
- recognised and phosphorylated by cis-Golgi kinases for binding to a mannose-6-P receptor in the trans-Golgi for sorting to the Late Endosome
- packaging into different uncoated CCVs with the same clathrin but AP1 adaptin molecule, which can also recognise the Man-6P receptor at the trans-Golgi, and converge with the endocytic cargo at the early endosome
What happens at the Late Endosome?
- lower PH causes dissociation of the Man-6P receptor
- produces molecules of ADP and Pi from ATP
- dephosphorylated cargo to produce a mature lysosomal hydrolase
- allows Man-6P receptor recycling back to the trans-Golgi
What happens at the lysosome?
proteases activate the hydrolases by proteolytic cleavage of the peptide bond, removing the protein tail that has been blocking its catalytic site, exposing it and releasing them.
Describe the basics of the SNARE hypothesis of 1993
- efficient and accurate targeted and compartmentalisation of vesicles
- introduced the concepts of the proteinous v-SNAREs and t-SNAREs
Describe SNAREs
- incorporated in the membranes and exposed on the cytoplasmic side of each vesicle (v) and target (t) membranes
- involved in determination of vesicle identity and targeting, as well as their corresponding NSF complex
Describe the specifics of the SNARE hypothesis of 1993
- different combinations of SNAREs exist for each vesicle targeting event, specific to their origins and destinations
- specificity of v/t-SNARE pairing provided the specificity required for vesicle targeting and fusion
Describe trans-SNARE formation
- tight trans-SNARE complex would be created upon docking, pulling the vesicle closer to the membrane for fusion and cargo release
- an NSF complex separates SNARE complices post-vesicle fusion in an ATP-dependent manner
- variant forms with unique identities of each of these proteins would be employed at each vesicle targeting step
Describe the SNARE hypothesis for compartments with mixed identities such as lipids and proteins
- vSNARE would have to be removed, docking onto the system and breaking it up
- separated SNAREs would be recycled, ready to transport and receive more cargo
Describe the infallibility programme of the SNARE hypothesis
wrongly targeted vesicles would approach a target membrane, but incompatibility would result in too much diffusion, temporally blocking it from fusion
