The Cell-39 Flashcards
The Secretory Pathway
How many proteins encoded the human genome are secreted?
15%.
They are known as the secretome.
How do proteins enter and leave the ER?
Enters the ER via protein translocation.
Leaves the ER via vesicular transport through the secretory pathway.
What does the ER do?
Lipid synthesis
Protein translocation (also in other organelles)
If proteins fail to fold/assemble, they get degraded. ‘Quality control’ point.
What is involved in protein translocation?
-Through a translocation pore.
-Proteins acquire their native structure. Folding + assembly.
-Proteins are N-glycosylated (the ER adds complex oligosaccharides onto certain amino acids)
How does protein translocation into the ER occur?
-Secretory proteins carry an N-terminal signal sequence which targets them to the surface of the ER.
-Protein translocation can happen as the protein is being translated (co-translational).
-Or after it has been translated with the help of chaperones (post-translational).
-In both cases, Sec61 is a core component of the eukaryotic translator.
-The signal sequence is removed once the protein is in the ER.
What makes the ER rough?
Polyribosome.
Each ribosome is docked on the ER membrane via a Single Recognition Particle (SRP) and an SRP receptor in the ER.
Many ribosomes can engage with a single mRNA molecule.
This leads to the docking of a ribosome-nascent chain complex onto the ER membrane giving the rER its ‘rough’ appearance.
Where do proteins move into the ER?
Through the lateral gate.
How is Sec61 a signal sequence-gated Aqueous Channel?
It has a ‘plug’ that allows it to be in a closed state.
And it is closed unless actively used to avoid diffusion of small molecules- that may change the concentration of ER lumen.
It opens for proteins containing an ER import signal sequence.
How does the signal sequence release the plug of Sec61?
The signal sequence is recognised by Sec61.
The signal wedges into a gate widening the central channel and releasing the plug.
Sec61 accommodates the segment of polypeptide following the signal sequence inside the channel.
The signal sequence (hydrophobic) leaves through the lateral gate and is cleaved off.
What are disulfide bonds regulated by?
PDI (protein disulfide isomerases)
What do chaperones do?
Detect protein misfolding: immature (in an intermediate state) or inappropriately folded, keeping proteins from aggregating and off the rest of the Secretory pathway.
What is the ER lumen full of?
Chaperones
Protein-folding catalysts
How are vesicles formed in a membrane bound organelle?
In a controlled way.
How does folding in the ER (N-glycosylation) take place? slide 10
How does vesicle budding work?
Vesicles work from a donor compartment and fuse with an acceptor compartment, allowing transport of luminal and membrane cargo.
The cell tightly controls what goes in and what is recycled in those vesicles so that organelles and environments can maintain their individual molecular identities.
What facilitates vesicle budding?
Vesicles are coated with proteins, this facilitates budding.
Different proteins coat vesicles at different stages of the secretory and endocytic pathways.
How are vesicles transported from ER to Golgi?
Once in the ER, proteins travelling towards the plasma membrane for secretion do not need signals for their sorting (they are secreted by default).
Therefore, enzymes heading for intracellular destinations (e.g. lysosome) must have a sorting signal to separate them from other proteins, and to sort them into the correct vesicles for transport to lysosomes.
What is the golgi complex?
Separate from the ER.
It is a stack of flattened membrane-enclosed sacs called cisternae (cisternal stack).
There are many vesicles around the rims of each cisterna.
What is the difference between the CGN and TGN?
CGN- cis Golgi network. Close to the ER.
TGN- trans Golgi network. Close to the rest of the secretory pathway.
Directional
What does the Golgi do?
Protein packaging and sorting:
-to the outside (secretion proper)
-to the plasma membrane
-to the lysosomes
Protein and lipid modification
-e.g. oligosaccharide trimming (CORE)
Oligosaccharide modifications of proteins act as signals for their folding and transport
How does transport to lysosomes take place?
-all lysosomal enzymes are glycoproteins, they have a sugar (glycan) attached to them as the enter the ER
-many of the mannose residues on the glycans become modified in the early Golgi to give mannose 6-phosphate: M6P acts as a targeting signal, for lysosome enzymes to move to lysosomes
-M6P receptors in the trans-Golgi membrane subsequently recognise M6P signals in the lysosomal enzymes
-binding is favoured at the pH of the trans-Golgi network (~6.5)
What are lysosomes?
Intracellular endpoint of secretory pathway.
The molecular dustbins of the animal cell.
Degrade/recycle exogenous particles/organisms.
Degrade/recycle old/exhausted proteins/organelles.
Rich in hydrolytic enzymes.
Low pH- lower than trans-Golgi vesicles.
What proteins characterise lysosomes?
Lysosomal enzymes- these are hydrolytic enzymes that work best at ~pH5. They include: proteases (e.g. cathepsins), nucleases, glycosidases, lipases, phospholipases and phosphatases.
What does clathrin do?
Clathrin forms cages around the vesicles in the trans-Golgi that are to move to the lysosomes.
The light and heavy chains form cages.
Give an overview of lysosomal transport?
Lysosomal proteins are modified with M6P.
M6P receptors bind M6P proteins in TGN at pH 6.5, allows for this binding to happen in an effective way.
M6P receptors recruit clathrin from the cytosol for vesicle budding.
The clathrin-coated vesicles becomes uncoated ready for fusion with late endosomes.
Late endosome low pH (5.5), causes the receptor-ligands dissociate.
Late endosomes fuse with lysosomes, delivering their cargo of enzymes.
Receptors recycles to the TGN via a different type of coated vesicle (the retromer coated vesicles).
What does the recognition of M6P signals by M6P receptors lead to?
A coating of part of the TGN membrane.
When ligands bind, the cytosolic tails of M6P receptors cluster and recruit cytosolic proteins to the membrane i.e. specific adaptor proteins (AP) and clathrin.
This clathrin/AP ‘coat’ deforms the membrane bud, then they can lose their proteins, so membrane is exposed.
Receptors are recycled back to golgi.
How do lysosomal enzymes get a M6P signal?
The lysosomal hydrolases are made, like secretory proteins, with ER signal peptides.
The signal peptides are cleaved in the ER, the proteins fold, and the proteins become glycosylated and then move into the golgi.
However, unlike other secretory proteins, these enzymes have a second (3D) targeting signal (a ‘signal patch’) that is recognised by an enzyme in the early golgi.
How does lysosomal hydrolase modify M6P?
Lysosomal hydrolase signal patch is recognised by the N-acetlyglucosamine phosphotransferase through its recognition site.
The catalytic site adds N-acetlyglucosamine phosphate.
A second enzyme removes the N-acetlyglucosamine leaving the M6P group.
What are lysosomal storage diseases?
Gaucher’s disease- lack of glucocerebrosidase, which breaks down glucocoerebroside, a cell membrane constituent of red and white blood cells.
I (inclusion) cell disease (mucolipidosis II)- multiple lysosomal enzymes are missing due to the absence of GlcNAc phospho-transferase, so undigested material, including muco-polysacharises, accumulate in lysosomes.
Tay Sachs disease- the hydrolase hexosaminidase A is not present in sufficient amounts in lysosomes leading to lysosomal accumulations of gangliosides in neurones.
Hunter and Hurler syndromes- hydrolases needed for breakdown of sulphated mucopolysaccharides are not present in sufficient amounts in lysosomes.
What controls folding of lysosomal hydrolases?
GlcNac phosphotransferase
Why can you not add a phosphate directly with an ATP-dependent kinase?
Recognition of the signal patch ensure lysosomal hydrolases are properly folded (and modified properly) before being sent off the lysosome. Diseases can occur as a result.
How can lysosomal malfunction occur?
If this signal patch or the golgi enzymes are mutated, these proteins do not end up with M6P signals. They are not then recognised by M6P receptors and don’t end up in the lysosomes (become secreted instead).
This means that material normally degraded by enzymes in lysosomes is no longer degraded leading to accumulation and lysosomal storage diseases.
