Secretory/Endocytic Pathways

Question 1

Give an overview of the entire pathway?

Answer 1

Endoplasmic Reticulum
Cis-Goligi network
Medial-Golgi network
Trans-Golgi network
Early endosome
Late endosome
Lysosome

Question 2

What are some general principals of the secretory/endocytic pathways?

Answer 2

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

Question 3

What proteins are need in membrane transport?

Answer 3

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

Question 4

Describe transport from endoplasmic reticulum to the Golgi?

Answer 4

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

Question 5

What are the theories on types of general transport between endoplasmic plasmic reticulum and the Golgi?

Answer 5

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

Question 6

ER to Golgi: how are COPII vesicles formed?

Answer 6

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

Question 7

ER to Golgi: how is cargo recruited into COPII vesicles?

Answer 7

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

Question 8

How are ER proteins retrieved from the Golgi?

Answer 8

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

Question 9

Describe the retrograde pathway of cargo proteins from the Golgi back to the ER?

Answer 9

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

Question 10

How do we retrieve ER membrane proteins from the Golgi?

Answer 10

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

Question 11

How do we retrieve ER luminal proteins from the Golgi?

Answer 11

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

Question 12

What is another theory of how ER proteins are retrieved from the Golgi?

Answer 12

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

Question 13

Describe the Golgi Apparatus?

Answer 13

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

Question 14

Describe protein modification in the Golgi?

Answer 14

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

Question 15

Describe sorting at the trans-Golgi network?

Answer 15

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

Question 16

How are cargo adaptor proteins regulated?

Answer 16

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

Question 17

What is used as a signal for trafficking to endosomes?

Answer 17

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

Question 18

Describe the mannose-6-phosphate receptor?

Answer 18

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

Question 19

After the Golgi what follows in the pathway?

Answer 19

Endosomes -> lysosomes

Endocytosis and exocytosis feeds into the lysosomal pathway

Question 20

What is the endocytotic pathway?

Answer 20

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

Question 21

Describe endocytosis of receptors?

Answer 21

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

Question 22

What are the types of endocytosis?

Answer 22

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

Question 23

What is clathrin mediated endocytosis?

Answer 23

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

Question 24

Describe the process of clathrin mediated endocytosis?

Answer 24

1. Endocytic coat proteins (from cytosol) cluster on the inner leaflet of the plasma membrane via adaptors: AP2, FCHO1 and CALM
2. The protein coat continues via further recruitment of other coat proteins
   E.g. Clathrin, scaffold proteins, epsins etc…
3. Cargo molecules concentrate at this coated region of the plasma membrane
4. This all promotes membrane bending - forms ‘clathrin-coated pit’
   As it imposes the lattice icosahedral cage of clathrin onto the membrane
5. 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
6. Actin polymerisation shapes the membrane of the vesicle
7. Disassembly releases endocytic machinery proteins and this uncoating releases the cargo - allowing for further trafficking in the cell
   This uses: chaperone HSC70 and dephosphorylation

Question 25

Describe endosomal sorting of LDL receptors?

Answer 25

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

Question 26

Describe trafficking to late endosomes/lysosomes?

Answer 26

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

Question 27

Describe exocytosis after the Golgi?

Answer 27

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

Question 28

What are some genetic diseases of the secretory/endocytic pathway?

Answer 28

Cranio-lenticulo-sutural dysplasia (CLSD)

Familial Hypercholesterolaemia

Question 29

Describe Cranio-lenticulo-sutural dysplasia (CLSD)?

Answer 29

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

Question 30

Describe Familial Hypercholesterolaemia?

Answer 30

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

Question 31

What are some pathogens/toxins that can manipulate the secretory/endocytic pathways?

Answer 31

Hepatitis C
Adenovirus
Cholera
Ricin

Question 32

Pathogens/toxins that can manipulate the secretory/endocytic pathways: Hepatitis C?

Answer 32

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

Question 33

Pathogens/toxins that can manipulate the secretory/endocytic pathways: Adenovirus?

Answer 33

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

Question 34

Pathogens/toxins that can manipulate the secretory/endocytic pathways: Cholera?

Answer 34

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

Question 35

Describe the action of the cholera toxin?

Answer 35

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

Question 36

Pathogens/toxins that can manipulate the secretory/endocytic pathways: Ricin?

Answer 36

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

Question 37

Describe the action of the ricin?

Answer 37

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