Secretory/Endocytic Pathways Flashcards
Give an overview of the entire pathway?
Endoplasmic Reticulum Cis-Goligi network Medial-Golgi network Trans-Golgi network Early endosome Late endosome Lysosome
What are some general principals of the secretory/endocytic pathways?
Many transport steps in the secretory and endocytic pathways involves the formation of vesicles that carry molecules to the target compartment membrane
Protein coats clathrin, COPI and COPII are used to form vesicles from the donor membranes and adaptors incorporate the cargo (proteins to be transported)
Once the vesicle is formed it uncoats
Transport through the secretory pathway is bidirectional - useful for recycling components
This maintains organelle homeostasis through retrograde membrane trafficking from the Golgi back to the ER
What proteins are need in membrane transport?
Rab proteins help bring the membranes together by binding to tethering proteins
SNARE proteins on the vesicle (v-SNARES) bind SNARE proteins on the target membrane (t-SNARES) and cause them to fuse thus delivering the cargo
When they interact the helical domains wrap around each other forming a stable 4 helix bundle = trans-SNARE complex, that locks the two membranes together
They ensure the correct membranes fuse together
Describe transport from endoplasmic reticulum to the Golgi?
Many of the proteins synthesised in the ER are exported via COPII vesicles to end up at their intended destination
Once formed these vesicles uncoat, they fuse together via SNARE interactions to form vesicular tubular clusters
Vesicular tubular clusters fuse with the cis Golgi network delivering the proteins to the Golgi apparatus
What are the theories on types of general transport between endoplasmic plasmic reticulum and the Golgi?
There is bulk flow of proteins from the ER to the Golgi as well as signal-mediated transport (cargo capture) through the secretory pathway
Bulk flow - cargo passively distributes between the donor compartment and the transport vesicles it generates = same cargo concentration within these two compartments
Signal-mediated transport - discrete export signals on the cargo are recognized and captured by specific receptors that are concentrated at sites of vesicle budding
However, there is a lack of any identified export signals on many of them - therefore they may reach their destination without the help of specific receptors
Partition in of the lipid bilayer - less often considered
ER to Golgi: how are COPII vesicles formed?
COPII coats are cage-like oligomeric lattices that drives vesicle formation
Sec12 catalyses GDP–GTP exchange on Sar1 causing it to bind to the ER membrane
Sar1 recruits Sec23–Sec24 to the ER membrane
Cargo is recruited into the forming vesicle by the adaptor Sec24 - as this recognises export signals located in the cytosolic tail of the cargo membrane
Sec13–Sec31 is recruited to complete the formation of the COPII coat and drive budding
ER to Golgi: how is cargo recruited into COPII vesicles?
Proteins require export signals for efficient export from the ER that are recognised by Sec24
Membrane proteins can be bound directly by Sec24, although some may require a receptor
Soluble proteins require a receptor that is recognised by Sec24
ERGIC-53 is the best characterized receptor and binds to soluble glycoproteins
ERGIC-53 has cytosolic a di-phenylalanine (FF) motif that acts as an export signal
NB proteins that lack export signals can also end up in COPII vesicles, including some ER resident proteins
How are ER proteins retrieved from the Golgi?
ER proteins can be non-selectively packaged in COPII vesicles
Many ER resident proteins have ER retrieval motifs that enable them to be retrieved from the Golgi
The ER export receptor ERGIC-53 is retrieved from the Golgi
These proteins can be incorporated into COPI vesicles are returned to the ER
Describe the retrograde pathway of cargo proteins from the Golgi back to the ER?
COPI heptamer is recruited to the Golgi by GTPase
This bends the membrane and binds to the cargo proteins
It does this via the recognition of discrete sorting signals on their cytosolic tails
It can then deliver the material back to the ER
In order for material to avoid immediate retrograde transport after anterograde transport there is a ‘valve’ like system between the ER-Golgi to prevent backflow of bulk-flow cargo
How do we retrieve ER membrane proteins from the Golgi?
The cytoplasmic tail of many ER resident membrane proteins has a dilysine KKXX ER retrieval motif
ERGIC-53 C-terminus is KKFF
Membrane proteins that have the KKXX motif that reach the Golgi are retrieved via interaction with the COPI coat complex
How do we retrieve ER luminal proteins from the Golgi?
Many soluble ER resident luminal proteins have a KDEL (Lys-Asp-Glu-Leu) retrieval motif e.g. BiP
The KDEL receptor binds the KDEL motif and is incorporated into COPI vesicles that transport proteins from the Golgi to the ER
Once in the ER the KDEL receptor dissociates from the ER protein and returns to the Golgi
What is another theory of how ER proteins are retrieved from the Golgi?
That the transmembrane domain of membrane proteins play a role in retrieval of ER proteins
Partitioning of TMDs into bilayer domains - linked with distinct physico-chemical properties
Within the early secretory pathway
Describe the Golgi Apparatus?
The Golgi apparatus is a complex organelle that comprises stacks of membranes (cisterna) that were first observed by Camillo Golgi in 1897
Best way to see it is electron microscopy
It is a major site of carbohydrate synthesis and sorts/dispatches products for the ER
It has tubular networks - entering at the cis-Golgi and exiting the trans-Golgi network
Contains two distinct membrane territories - that comprise the early and distal secretory pathways, respectively
Thin - cholesterol/sphingolipid bilayers with loosely packed lipids and neutral cytoplasmic surface charge
Thick - cholesterol/sphingolipid rich bilayers with tightly packed lipids and a negative cytoplasmic surface charge
Contains membrane proteins containing transmembrane domains (TMDs) matches the thickness of the membrane territory it resides in
Newly synthesized proteins from the ER traffic through the Golgi apparatus in a cis to trans direction
Describe protein modification in the Golgi?
Glycosylation has multiple roles: solubility, protection from degradation by proteases and molecular recognition
In the Golgi N-linked oligosaccharides are modified by removal of 3 mannoses
O-linked glycosylation is catalysed by glycosyl transferase enzymes
Further modifications can occur to produce complex oligosaccharides, but if the oligosaccharides are inaccessible this results in high mannose oligosaccharides
We can produce proteoglycans and glycosaminoglycans for example
Glycosylation promotes protein folding - as intermediates are mores soluble preventing aggregation and mediates binding of protein chaperones
Describe sorting at the trans-Golgi network?
The trans-Golgi network (TGN), acts as a sorting station:
Proteins to be targeted to the plasma membrane secretion require no specific signals
Tubules form from the TGN that subsequently fuse with the plasma membrane
Trafficking to endosomes requires specific signals and involves formation of clathrin coated vesicles
Proteins can also be sorted to apical/basolateral plasma membrane, endosomal compartments and specialised secretory organelles e.g. granules in endocrine cells
Cytosolic cargo adaptor molecules are the main players in the sorting process
They recruit clathrin and contain 4 subunits
At the C-terminal domain they recognise cargo proteins containing a tyrosine motif
How are cargo adaptor proteins regulated?
Protein sorting requires the recruitment of cytosolic cargo adaptors and the binding of these adaptors to cargo molecules
This is regulated by small GTPases of the Arf family (ADP-ribosylation factor) and phospholipids
Small GTPases, cause an N-terminal helix to become exposed and inserted into the lipid bilayer, inducing membrane deformation and facilitating formation of vesicles/recruitment of cargo adaptors
Some cargo proteins bind directly to phospholipids e.g. Phosphatidylinositol-4-phosphate and phosphatidylserine
What is used as a signal for trafficking to endosomes?
Mannose-6-phosphate - a sorting signal added to newly synthesised N-linked oligosaccharide lysosomal hydrolases - in the Golgi
M6P is a targeting signal for the endosomal pathway recognised by M6P receptors (MPRs) at the TGN used to sort proteins to endosomes
Arf-binding proteins (clathrin associated cargo adaptors) bind to the M6P receptors and incorporate these into clathrin-coated vesicles via the adaptor protein complex AP-1
Arf1 is an allosteric activator of AP-1
Epsin-related proteins can also be used as a clathrin adaptor - to recruit clathrin and regulate traffic between the TGN and endosomes
M6P receptors cycle between the trans-Golgi network and endosomes
Describe the mannose-6-phosphate receptor?
Two types: cation-dependent and cation independent
The luminal domain contains 15 homologous repeat domains - each domain containing 150 amino acids
There are 2 binding sites at domain 3 and 9
Residues adjacent to domain 3/5 facilitate binding to phosphomannosyl residues
Domain 11 has a binding site for insulin-like growth factor II = helps clear IGF II, by delivering to lysosomes for degradation
After the Golgi what follows in the pathway?
Endosomes -> lysosomes
Endocytosis and exocytosis feeds into the lysosomal pathway
What is the endocytotic pathway?
Endocytosis is the uptake of molecules from the plasma membrane and extracellular material into the cytoplasm of the cell
Plasma membrane derived vesicles deliver endocytosed molecules to early endosomes
Early endosomes are sorting stations:
Recycling back to the plasma membrane via recycling endosomes and trafficking to lysosomes
Early endosomes form multivesicular bodies which via late endosomes deliver material to lysosomes for degradation
Early endosomes - late endosome - endolysosome - lysosome
Describe endocytosis of receptors?
Plasma membrane receptors can capture molecules for uptake into cells e.g.
Low-density lipoprotein (LDL) receptors take up LDLs which carry cholesterol
LDL receptors release their ligands in early endosomes and traffic back to the cell surface
Signalling receptors bind ligands and can be down-regulated by endocytosis to turn off their signalling e.g.
Epidermal growth factor (EGF) receptors binding to EGF activates signalling pathways
EGF binding also triggers endocytosis and trafficking of the receptor to lysosomes to turn off signalling - drives the proliferation
What are the types of endocytosis?
There are multiple forms of endocytosis: clathrin mediated, caveolae mediated, macropinocytosis, phagocytosis
Clathrin is the most common
A retromer (not basket shaped vesicle) can also form
What is clathrin mediated endocytosis?
Clathrin mediated endocytosis involves pinching off the plasma membrane by forming clathrin coated vesicles
A clathrin subunit is a triskelion - which assemble to form a basket like structure
Proteins can be recruited into the clathrin coated vesicles via adaptors (AP-2 and others)
LDL and activated EGF receptors can all be endocytosed by clathrin coated vesicles
There is a threshold of cargo within the vesicle budding process; if not reached it can be aborted or delayed
Describe the process of clathrin mediated endocytosis?
- Endocytic coat proteins (from cytosol) cluster on the inner leaflet of the plasma membrane via adaptors: AP2, FCHO1 and CALM
- The protein coat continues via further recruitment of other coat proteins
E.g. Clathrin, scaffold proteins, epsins etc… - Cargo molecules concentrate at this coated region of the plasma membrane
- This all promotes membrane bending - forms ‘clathrin-coated pit’
As it imposes the lattice icosahedral cage of clathrin onto the membrane - The neck of the membrane invagination constricts and is cut sing dynamin/GTP to separate the clathrin-coated vesicle away from the plasma membrane
This is mediated by the BAR domain - Actin polymerisation shapes the membrane of the vesicle
- Disassembly releases endocytic machinery proteins and this uncoating releases the cargo - allowing for further trafficking in the cell
This uses: chaperone HSC70 and dephosphorylation
Describe endosomal sorting of LDL receptors?
Endocytosed LDL receptors release their ligands in the acidic early endosome
LDL receptors recycle back to the plasma membrane via vesicles that either fuse with recycling endosomes or directly with the plasma membrane
The LDL receptors are then available again to capture ligands
LDL receptors can cycle from plasma membrane to early endosomes and back again in 10 min
Describe trafficking to late endosomes/lysosomes?
Epidermal growth factor binding by epidermal growth factor receptors activates signalling cascades
Epidermal growth factor receptors are downregulated when they bind the epidermal growth factor to turn off signalling
The ubiquitinated receptor is endocytosed by clathrin mediated endocytosis, trafficked to early endosomes and then taken into a multivesicular body by invagination - preventing further signalling
Multivesicular bodies are converted into late endosomes which fuse with lysosomes where the receptors are degraded
Therefore multivesicular bodies help transport for degradation
Describe exocytosis after the Golgi?
Three classes of protein leaving the trans-Golgi:
Destined for lysosomes, secretory vesicles and immediate delivery to the cell surface
Secretory vesicles wait near the plasma membrane until signalled to release their contents e.g. Hormone
When it fuses to the membrane the contents are removed by endocytosis almost as quickly as the endocytosis - therefore only transiently increases the plasma membrane
What are some genetic diseases of the secretory/endocytic pathway?
Cranio-lenticulo-sutural dysplasia (CLSD)
Familial Hypercholesterolaemia
Describe Cranio-lenticulo-sutural dysplasia (CLSD)?
Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterised skeletal defects, facial abnormalities
CLSD is caused by mutations in Sec23a, a component of the COPII machinery
The F382L SEC23A mutation results in:
Dilation of the ER
Reduced capacity to generate cargo-containing vesicles
Reduction in collagen export from the ER
Describe Familial Hypercholesterolaemia?
Disruption of LDL receptor endocytosis can cause Familial Hypercholesterolaemia by reducing LDL removal from the blood and cholesterol deposits in the walls of arteries
The JD mutation in the LDL receptor is in the NPVY sequence motif required to recruit LDL receptor into clathrin coated vesicles
Mutations can also affect the adaptor Autosomal Recessive Hypercholesterolemia (ARH) protein, an adaptor which binds the LDL receptor NPVY sequence
What are some pathogens/toxins that can manipulate the secretory/endocytic pathways?
Hepatitis C
Adenovirus
Cholera
Ricin
Pathogens/toxins that can manipulate the secretory/endocytic pathways: Hepatitis C?
Hepatitis C virus - a single-strand RNA virus that targets hepatocytes, through a multistep complex entry
E2 domain within the glycoprotein envelope of HCV binds to the CD81 receptor complex on the cell surface, which HCV uses as receptor to attach and enter hepatocytes
This is a major cause of liver cirrhosis and hepatocellular carcinoma
HCV uses clathrin mediated endocytosis and requires the AP-2 adaptor protein complex to enter the cell
Once endocytosed it traffics to early endosomes prior to releasing the viral genome into the cytosol
Potential HCV entry inhibitors include: antibodies targeting the viral envelope or siRNA against the host cell factors
Pathogens/toxins that can manipulate the secretory/endocytic pathways: Adenovirus?
This hijacks the ER retrieval pathway to inhibit MHC class I trafficking Adenovirus E3/19K is an integral membrane protein that binds MHC class I via its luminal domain The cytoplasmic tail of E3/19K has a dilysine ER retrieval motif (KKXX) that enables E3/19K /MHC class I to be retrieved from cis Golgi via interaction with the COPI complex This prevents MHC class I from reaching the plasma membrane
Pathogens/toxins that can manipulate the secretory/endocytic pathways: Cholera?
Cholera toxin is secreted by Vibrio Cholerae and is a severe diarrheal disease
Most prevalent in the third world, cholera was a major problem in the UK before the introduction of proper sanitation
Cholera toxin promotes electrolyte and water movement into the intestinal lumen resulting in severe diarrhoea and further spread of the bacterium
Describe the action of the cholera toxin?
Cholera toxin is 2 subunit protein
The B subunit (CTB) binds to the GM1 glycolipid on the surface of cells that line the gut
CTA1 once in the cytosol activates adenylate cyclase increasing cAMP, which in turn activates protein kinase A (PKA)
PKA phosphorylates CFTR promoting Cl- secretion and therefore osmotic movement into the intestinal lumen
CTA1 is unfolded before transport to cytosol - but it takes the advantage of the ERAD pathway to accomplish retrotranslocation
It has low lysine content - protects it from degradation of ERAD
Pathogens/toxins that can manipulate the secretory/endocytic pathways: Ricin?
Ricin is a plant toxin that is present in castor oil seeds and can be extracted from this source for use as a poison
The symptoms of ricin poisoning include: Nausea, Diarrhoea, Seizures and Hypotension
Ricin was used to kill Georgi Markov, a Bulgarian dissident in 1978
Describe the action of the ricin?
Ricin is a 2 subunit protein (heterodimeric), both 30 kDa linked by a di-sulfide bond
The A-chain is enzymatically active and B-chain binds to glycolipids and glycoproteins on the surface of cells
Ricin is endocytosed and is trafficked via the TGN to the ER, although it lacks a KDEL sequence
In the ER the A-chain dissociates from the B-chain, is unfolded and retrotranslocated across the ER membrane into the cytosol
Once in the cytosol the A-chain escapes proteosomal degradation
The cytosolic target for the A-chain is the 28S ribosomal RNA
The A-chain depurinates it by cleaving the bond between an adenine residue (A4324) and ribose releasing the adenine
This corresponds to the site for elongation factor binding, which can no longer bind hence inhibiting protein translation
