Lecture 8 trafficking 3 Flashcards
Why do SNAREs required cooperation with Rab proteins?
For accurate vesicle docking
How many family members are in Rab?
60 known
What type of proteins are Rabs?
Monomeric GTPases. Do not polymerise.
Where are they located?
Both cytosolic (non active form) and membrane associated fractions (active form).
What is the GDP bound form?
Soluble and inactive.
What is the GTP bound form?
Membrane associated and active.
Where do each Rab proteins predominantly localised (not black and white)?
Rab1=ER and golgi complex
Rab2= cis golgi network
Rab3A=synaptic vescicles, secretory granules
Rab4=early endosomal
Rab5A=plasma membrane, clathrin-coated vesicles
Rab6=medial and trans Golgi network
Rab7=late endosomes
Rab8=secretory vesicles (basolateral)
Rab9=late endosomes, trans Golgi network
Useful experimentally as if find rab1 for example you know you are looking at the ER.
How were the protein localisations found?
Their domains were swapped with eachother e.g., Rab7 c terminus swapped with Rab1 c terminus. Shows different localisation.
What are the steps to Rab vesicle trafficking?
- Rab activation. GEF converts Rab-GDP to Rab-GTP. The lipid group on Rab allows it to associate with the donor membrane.
- The Rab-GTP associates with a vesicle along with R-SNARE proteins. The vesicle buds off from the donor compartment and moves towards the target membrane.
- The Rab-GTP interacts with Rab effectors at the target membrane, assisting vesicle docking. The R-SNARE on the vesicle aligns with the Q-SNARE on the target membrane.
- The SNARE complex mediates membrane fusion, allowing cargo delivery. Rab-GTP undergoes hydrolysis to Rab-GDP, triggered by GAP.
- The Rab-GDP is extracted from the membrane by GDP dissociation inhibitor GDI.
Why are the Rab effectors big and fibrilar?
Extend far away from the target compartment so the first interaction is the Rab and effector before relevant SNAREs. Not sure why this is needed?
How are Rab proteins modified?
PTMs lipids that are hydrophobic. Similar to ARF and SAR1. E.g., Rab5. RABEX5 is a GEF for Rab5 particular to Rab.
How does rab effector and rab binding increase specificity?
Has a limited number of possible bindings. Can only dock and fuse at correct compartments.
How do rab effectors themselves localise to the correct compartment?
They contain localisation domains. For example, EEA1 and Rebenosyn 5 contain FYVE domains. Zinc ions bind to FYVE domains to facilitate correct structures. Also have a loop that contains hydrophobic residues.
How is PIP3 involved?
PIP3 is enriched in early endosome membranes. FYVE domains interact with them so the effectors are localised. The loop can then insert itself into the membrane to stabilise the interaction.
How does FYVE domain only interact with the PIP3 and not other PIPs?
Absolutely conserved with basic motifs. The nitrogens on the histidine side chain of FYVE domain can interact with the phosphate head groups on PIP3 due the the steric spacing on PIP3. E.g., PIP4 has a steric clash with the conserved histidine.
What else can Rab5 do?
Recruit other proteins such as PIP kinase to form PIP3 to recruit Rab effectors.
How do Rab 5 and 7 act in vesicular maturation?
Mon1/Ccz1 recruits Rab7 and activates it. Rab7 recruits TBC1D18 which is a GAP. GAP is active on Rab5 so stimulates GDP bound Rab5. Loses its Rab5 functionality and gains Rab7 functionality this is the transition from early to late endosome.
How does the cell ensure complete proper folding of proteins to exit the ER?
An unfolded protein gets modified by a mannose structure which gets glucose moieties added. Glucose trimming in ER leaves mannose with one glucose. Calnexin recognises this (membrane bound ER chaperone). Then releases the protein if correctly folded recruited by COPII to leave. If not the glucose moiety is trimmed off by a transferase enzyme. The cycle repeats. If the protein cannot fold properly it gets degraded.
How does retention of IgG in the ER for correct folding occur an example of a quaternary structured molecule?
When the heavy chains aren’t assembled properly they have exposed hydrophobic residues. These are bound to BiP (Hsc70 chaperone family). The heavy chains can’t be recruited by COPII. As they antibody light chains are assembled to the heavy chains they block BiP from attaching. When formed no BiP so the protein can leave the ER.
How does degradation of unfolded proteins occur?
The core mannose structure gets modified so abnormal. Recognised by lectin. Retrotranslocated out of ER through sec61. Enzymes involved in ubiquitination such as E3 ligase. An ATPase moves the protein away from the ER. N-glycanase removes sugars from the protein. It goes to the proteosome.
How does the trafficking system distinguish resident ER proteins from others?
ER retrieval signals. Identify small conserved motifs. ER membrane proteins e.g., KDEL receptor has 4 C terminal lysines with two other amino acids e.g., KKXX. ER lumen protein such as BiP contain a KDEL sequence. KDEL receptors have different affinities in different compartments for retrieval. This is based upon pH. KDEL receptors have high affinity in golgi.
Why do golgi enzymes not have internal signals?
Not sure why.
How do proteins stay in the right compartment?
KIN recognition. Resident proteins in the compartment can join together to form a super molecular protein complex so they are too big to leave.
How are membrane proteins retained in compartments e.g., golgi?
By shorter transmembrane domains than plasma membrane proteins.
How is glycosylation important for localisation and function?
- ERGIC53 proteins recognise high mannose proteins.
- The Notch protein requires specialised glycosylation to signal correct cell fate.
- Mannose-6-phosphate controls transport into the lysosome.
How does recognition of M6P addition occur?
Lysosomal hydrolases carry M6P targetting moieties.
How does the golgi recognise when to add an M6P moiety?
Signal for M6P addition must reside in the primary sequence. When the hydrolase is mature the tertiary structure produces a characteristic signal patch which is recognised by an acetly glucosamine phosphotransferase enzyme. Has maturing M6P moiety.
What are the steps to M6P tagging pathway?
The lysosomal enzyme has an N-linked oligosaccharide with terminal mannose residues. A signal patch on the enzyme is recognized by GlcNAc phosphotransferase.
The enzyme UDP-GlcNAc (Uridine Diphosphate N-Acetylglucosamine) binds to the catalytic site of the GlcNAc phosphotransferase.
The GlcNAc-P (N-Acetylglucosamine phosphate) is transferred to a mannose residue on the lysosomal enzyme.
This phosphorylation marks the enzyme for lysosomal targeting.
The enzyme with GlcNAc-P attached to mannose is released from the phosphotransferase.
The GlcNAc group is removed, leaving a mannose-6-phosphate (M6P) tag.
The M6P tag allows the enzyme to be recognized by M6P receptors in the trans-Golgi network.
The tagged enzyme is then transported to lysosomes, where it becomes functional.
What are the different types of MPRs? Mannose 6 phosphate receptors?
Cationic dependent MPR in golgi. Cationic independent found in golgi and cell surface.
How does cationic independent protein get used in research?
as they are present on the cell surface lysosomal hydrolase can be injected and transported in through cationic independent proteins.
