Lecture 39: The Secretory Pathway Flashcards
Monday 20th January 2025
What percentage of proteins encoded by the human genome are secreted?
15%
How do proteins enter the Endoplasmic reticulum?
Proteins enter the ER via protein translocation and leave the ER via vesicular transport through the secretory pathway
What does the ER do?
- Lipid synthesis
- Protein translocation (also in other organelles):
- through a translocation pore
- proteins acquire their native structure, folding and assembly
- proteins are N- glycosylated (the ER adds complex oligosaccharides onto certain aminoacids)
- If proteins fail to fold/assemble, they get degraded, quality control point
Describe protein translocation into the ER
- 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 translocator
- The signal sequence is removed once the protein is in the ER
Describe co-translational protein translocation into the ER
- Each ribosome is docked on the ER membrane via a Signal Recognition Particle (SRP) and an SRP receptor in the ER
- This allows proteins to move to the inside of 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
Describe Sec 61
- Sec 61 is 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
- It opens for proteins containing an ER import signal sequence
Describe how the ER import signal sequence wedges in Sec61 and releases the plug
- 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
Describe protein folding in the ER (chaperones and disulfide bonds)
- ER lumen is full of chaperones and protein-folding catalysts
- Chaperones detect protein misfolding (immature or inappropriately folded proteins) keeping proteins from aggregating and off the rest of the Secretory pathway
- Disulfide bonds are regulated by PDI (Protein Disulfide Isomerases)
What are protein chaperones?
- Protein chaperones are specialized proteins that assist other proteins in folding correctly, preventing misfolding and aggregation.
- They do not become part of the final protein structure but help stabilize unfolded or partially folded proteins, ensuring they reach their proper three-dimensional shape.
What are disulfide bonds regulated by?
PDI
What does PDI do to disulfide bonds?
PDI reduces disulfide bonds
Is it true that chaperones bind to mis-folded proteins and keep them from aggregating?
yes
Protein folding in the ER (N-glycosylation)…
- As soon as the proteins enter the ER lumen, they are N-glycosylated in some Asparagine (Asn) residues
- The precursor oligosaccharide is modified (trimmed) for proper protein folding
- The core structure is further modified in the Golgi
After the ER, what pathway do proteins enter?
They enter the secretory pathway
Give a one secntence summary of the secretory pathway
Vesicle movement and intracellular trafficking
Describe vesicle budding of the secretory pathway
- Vesicles bud from a donor compartment and fuse with an acceptor compartment (e.g. ER to early Golgi, from Golgi to late endosomes etc..)
- This allows for the 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
In the secretory pathway, what are vesicles coated with?
They are coated with proteins
Why are the vesicles of the secretory pathway coated with proteins?
Because this facilitates budding
Is it true that different proteins coat vesicles at different stages of the secretory and endocytic pathways?
Yes
Once in the ER, do proteins travelling towards the plasma membrane for secretion need signals for their sorting?
No, they are secreted by default.
Why must enzymes/proteins heading for intracellular destinations, such as lysosomes, have a sorting signal?
- To separate them from other proteins, and to sort them into the correct vesicles for transport to lysosomes
Describe 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 does the golgi do?
- Packages and sorts proteins to: the outside, the plasma membrane, the lysosomes.
- Modifies proteins and lipids (e.g. Oligosaccharide trimming).
What do oligosaccharide modifications of proteins act as ?
Oligosaccharide modifications of proteins act as signals for their folding and transport
Describe the secretory pathway – Transport to lysosomes
- All lysosomal enzymes are glycoproteins: they have a sugar (the glycan) attached to them as they 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
- 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)
Describe 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
What proteins characterise lysosomes?
- lysosomal enzymes - these are hydrolytic enzymes that work best at ~pH 5.0. They include:
- proteases (e.g. cathepsins)
- nucleases
- glycosidases
- lipases
- phospholipases
- phosphatases
Recognition of M6P signals by M6P receptors leads to a coating of that part of the Trans-Golgi Network 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 into a bud.
Is it true that Clathrin forms cages around the vesicles in the trans-Golgi that are to move to the lysosomes?
Yes
Give an overview of lysosomal transport
- Lysosomal proteins are modified with M6P
- M6P receptors bind M6P proteins in TGN (Trans-Golgi Network) at pH 6.5
- M6Preceptors 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 recycle to the TGN via a different type of coated vesicle (the retromer coated vesicle)
cis FACE =
ER
trans FACE =
rest of secretory pathway
Is it true that all lysosomal enzymes have a M6P targetting signal which bind to M6P receptors?
Yes
How do lysosomal enzymes get an M6P signal?
- The lysosomal hydrolases are made, like secretory proteins, with ER signal peptides that help direct them to the ER for proper folding and processing.
- The signal peptides are cleaved in the ER, the proteins fold, and the proteins become glycosylated
- However, unlike other secretory proteins, these enzymes have a second (3 dimensional) targeting signal (a ‘signal patch’) that is recognised by an enzyme in the early Golgi.
- Lysosomal hydrolase signal patch is recognised by the N-acetylglucosamine phosphotransferase though its recognition site
- The catalytic site adds N-acetylglucosamine phosphate
- 3 A second enzyme removes the N-acetylglucosamine leaving the M6P group
What controls the folding of lysosomal hydrolases?
GlcNac phosphotransferase
Why not adding a phosphate directly with an ATP-dependent kinase?
Recognition of the signal patch ensures lysosomal hydrolases are properly folded before being sent off to the lysosome
What happens if the signal patch or the golgi enzymes are mutated?
- 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
What are some lysosomal storage diseases?
- Gaucher’s disease
- I (inclusion) cell disease (mucolipidosis II)
- Tay Sachs disease
- Hunter and Hurler syndromes
Describe Gaucher’s disease
lack of glucocerebrosidase (lysosomal acid β-glucosidase), which breaks down glucocerebroside, a cell membrane constituent of red and white blood cells.
Describe I (inclusion) cell disease
multiple lysosomal enzymes are missing due to the absence of GlcNAc phospho-transferase, so undigested material, including muco-polysaccharides, accumulate in lysosomes
Describe Tay Sachs disease
the hydrolase hexosaminidase A is not present in sufficient amounts in lysosomes leading to lysosomal accumulations of gangliosides in neurones
Describe Hunter and Hurler syndromes
hydrolases needed for breakdown of sulphated mucopolysaccharides (e.g. iduronidase that breaks down glycosaminoglycans – next lecture) are not present in sufficient amounts in lysosomes
Summary
- The ER imports proteins from the cytosol via protein translocation
- The secretory pathway is a functional array of membrane-bounded organelles - Interconnected via vesicle trafficking
- Proteins need a signal peptide to enter the secretory pathway and be secreted
- To reach the lysosomes, they need an extra signal, M6P. Yet another signal (a 3D patch) is required for lysosomal proteins to acquire the M6P
- Any defect in lysosomal sorting leads to major diseases
Is clathrin an example of a vesicle coating protein?
Yes
The cis face of the golgi appartus receives proteins from the ER.
The trans face of the golgi apparatus ships proteins to final destinations.
The cis face of the golgi appartus receives proteins from the ER.
The trans face of the golgi apparatus ships proteins to final destinations.
Does the golgi apparatus further modify oligosaccharides?
Yes
Lysosomal enzymes are glycoproteins tagged with mannose-6-phosphate (M6P).
The mannose-6-phosphate (M6P) is added to the lysosomal enzymes via a two-step enzymatic process, which is…
- GlcNAc-phosphotransferase adds GlcNAc-phosphate to mannose
- Second enzyme removes GlcNAc, exposing M6P tag
M6P Recognition summary
M6P receptors in trans-Golgi bind tagged enzymes at pH ~6.5
Receptors recruit clathrin and adaptor proteins → vesicle formation
Vesicles fuse with late endosomes/lysosomes
Lower pH (~5) causes receptor to release enzyme
Receptors are recycled back to Golgi via retromer-coated vesicles
What is the entry point for proteins into the secretory pathway?
The endoplasmic reticulum
Is is true that proteins must be properly folded and glycosylated to proceed in the secretory pathway?
Yes
What enables translocation?
Sec61
What is critical for lysosomal targetting?
The M6P tag
