Block C Lecture 1 - Introduction to Protein Sorting and the Secretory Pathway Flashcards
What does cell maintenance and growth require?
New protein and lipid synthesis, as well as the ability to target these to their correct membrane component (wherever their function is required)
(Slides 4 and 5)
What is cell maintenance, growth and the uptake / secretion of molecules mediated by?
Via “membrane traffic” or “vesicle transport”
(Slide 4)
What are the 5 locations which the secretory pathway mediates protein sorting to?
The cell exterior (secreted proteins)
Plasma membrane
Lysosomes
Golgi
Endoplasmic reticulum
(Slide 6)
What are the 4 basic steps of the secretory pathway?
- Protein is imported into ER
- ER to Golgi apparatus vehicle transport
- Intra Golgi transport
- Vesicle transport to either the plasma membrane or the lysosomes
(Slide 7)
What are the 2 key steps of vesicle transport and what is each of these steps mediated by?
The vesicle buds from a “donor” membrane compartment e.g ER. Vesicle budding is mediated by coat proteins.
The bud is then accepted by an “acceptor” membrane compartment e.g Golgi. To do this the vesicle needs to fuse to the compartment. This is mediated by SNARE proteins
(Slide 8)
What do coat proteins recruit?
“Cargo” proteins
(Slide 9)
What are the functions of vesicles mediated by the COPI, COPII and clathrin coat protein complexes?
COPI Vesicles function in retrograde movement from the Golgi to the ER
COPII vehicles move proteins from the ER to the Golgi
Clathrin-coated vesicles function in endocytosis
(Slide 9)
Why is the retrograde movement of proteins from the Golgi to the ER via vesicles mediated by COPI coat proteins important?
For ER retainment of some proteins
(Slide 9)
What are the 2 type of SNARE proteins which mediate vesicle fusion with the acceptor membrane compartment, and what are 2 other proteins involved?
vehicle SNAREs (VSNAREs)
SNARE proteins present on thee target membrane (known as tSNAREs)
Also involved:
Tethering proteins and Rab proteins
(Slide 10)
What is protein translocation across the ER membrane mediated by?
Signal sequences
(Slide 12)
Protein translocation into the ER occurs co-translationally. What does this mean?
It means it occurs as the protein is still being synthesised
(Slide 13)
What are the steps of protein translocation into the ER?
- A “signal sequence” is recognised by Signal Recognition Particle (SRP)
- SRP halts protein translation and takes the ribosome to the ER
- The signal sequence then inserts into a translocation channel on the ER membrane and protein synthesis resumes
- As the protein is synthesised, it gets threaded through a channel into the ER
- When synthesis is complete, the signal sequence is cleaved from the protein
(Slide 13)
Where are signal sequences (signal peptides) present on a protein and what do they contain?
They are present on the N-terminus and contain a stretch of hydrophobic amino acids (usually 9-12 hydrophobic residues)
(Slide 14)
What are hydrophobic stop-transfer signals used for?
To target the ER
(Slide 15)
What happens when a protein with a hydrophobic stop-transfer signal interact with the ER?
They interact with the translocation channel, causing a conformational change in the channel, and discharging the protein laterally (forwards) into the membrane, with the stop-transfer signal becoming a trans-membrane domain
(Slide 15)
What protein topology (orientation) do cleavable signal peptides always give rise to?
The N-terminus residing inside the ER
(Slide 15)
How does the topology of proteins with start-transfer sequences determined by charge?
If there are more positively-charged amino acids preceding then following the hydrophobic core of the start-transfer sequence then the N-terminus resides in the cytoplasm
If there are less then the C-terminus resides in the cytoplasm
(Slide 16)
What is the difference between a start-transfer and a stop-transfer sequence?
A start-transfer sequence initiates insertion into the ER membrane and/or translocation into the ER lumen whereas a stop-transfer sequence halts translocation into the ER lumen and anchors the protein in the membrane
(Slides 15 and 16)
Do all proteins that have a start-transfer sequence have a stop-transfer sequence and vice-versa?
No, a protein can have either one of these, or both
(Slides 15 and 16)
What is a polytopic protein?
A protein which crosses the membrane multiple times
(Slide 17)
How do polytopic proteins use multiple start and stop-transfer sequences to be threaded through the ER membrane multiple times?
The first start and stop transfer sequences essentially result in 1 end (N-terminus) being in the cytoplasm. Then repeating start and stop transfer sequences start and halt translocation, and inserts another transmembrane domain.
(Slide 18)
What is the role of chaperone proteins in the ER?
They bind to unfolded translocated proteins and assist in their correct folding
(Slide 20)
What does the chaperone BiP (Binding protein) do specifically?
It binds to exposed hydrophobic regions of proteins, preventing aggregation and allowing proteins to fold correctly, and also prevents translocated polypeptides from moving from the ER back into the cytosol
(Slide 20)
When does protein glycosylation begin?
When the protein is embedded into the ER lumen
(Slide 21)
What occurs during protein glycosylation in the ER?
The oligosaccharide is removed from a dolichol lipid carrier and is transferred to the polypeptide chain during their translocation across the ER membrane
(Slide 21)
What does N-linked glycosylation involve?
The attachment of sugar groups onto asparagine residues (N is the single letter code for asparagine)
(Slide 21)
What are 2 examples of functions which protein glycosylation can have?
It can help protein folding or regulate protein targeting
(Slide 21)
How does the ER act as a protein quality control system?
As incorrectly folded proteins are targeted for degradation via ERAD (ER-associated protein degradation)
(Slide 22)
What occurs in ERAD (Er-associated protein degradation)
A mis-folded protein is retrotranslocated through the ER membrane, ubiquitinated and degraded by proteasome
(Slide 22)
What occurs after proteins are translocated into the ER and pass quality control?
They are translocated across the ER membrane (both membrane and soluble proteins) and either moved by vesicle transport to the next compartment of the Secretory Pathway (the Golgi) or they are retained at the ER if their function is required there
(Slide 23)
What is the difference between membrane and soluble proteins?
Membrane proteins get imbedded in the ER membrane (they have both a start and stop-transfer sequence)
Soluble proteins fully enter the ER (they only have a start-transfer sequence)
What is the KDEL sequence?
A retrieval sequence which is only present on soluble ER proteins
(Slide 24)
Where does the KDEL sequence get it’s name from?
It’s composed of 4 amino acids: Lysine, Aspartic acid, Glutamic acid and Leucine, with their single letter codes spelling out KDEL
(Slide 24)
How does the KDEL sequence work?
- The KDEL sequence is recognised by a KDEL receptor located in the Cis Golgi or the ER.
- When proteins with the KDEL sequence accidentally move from the ER to the cis Golgi, the KDEL receptor binds these proteins
- It then directs them back to the ER using vesicles mediated by COPI proteins
- KDEL receptor affinity depends on pH level, with it having a higher affinity at a lower pH. So when the protein gets transported back to the ER, the higher pH results in the KDEL receptor releasing the protein
(Slide 24)
What are the cis and trans Golgi?
The cis Golgi side is the side which is closest to the ER whereas the trans Golgi side faces the plasma membrane
(Slide 24)
What happens in the Golgi?
Glycosylation sequences created in the ER undergo trimming and modification by other sugars
(E.g. removing mannose residues, adding N-acetylglucosamine, galactose or other residues)
(Slide 26)
Why is mannose-6-phosphate glycosylation a special kind of glycosylation?
As proteins glycosylated with this are recognised by the mannose-6-phosphate receptor and enter transport vesicles which target them to lysosomes
(Slide 27)
What is an example of a protein which is targeted to lysosomes and why are they targeted to the lysosomes?
Hydrolytic enzymes (hydrolases) - targeted to the lysosome as they are the site of protein degradation
(Slide 27)
What are 2 diseases which defects in protein sorting is linked with?
Infantile neuronal ceroid lipofuscinosis (a neurodegenative disorder)
Cystic fibrosis
(Slide 29)
How can defects in protein sorting result in infantile neuronal ceroid lipofuscinosis?
It’s caused by mutations in a lysosomal enzyme which prevents its movement from the ER, leading to lysosomal dysfunction
(Slide 29)
How can defects in protein sorting result in cystic fibrosis?
Some mutations in the CFTR gene (encodes a chloride channel) can lead to a loss of targeting to the plasma membrane via the secretory pathway. This impairs transport of chloride ions across the membrane and out of the cells and can impair the movement of water in and out of cells.
This results in cells which line the passageways of the lungs, pancreases and other organs, producing mucus that is abnormally thick and sticky
(Slide 29)
