Lecture 4 - Protein Targeting Flashcards
Why is Protein Targeting Important?
Cells contain multiple compartments, therefore proteins must be targeted from the main compartment (cytosol) to their home compartment to allow their proper function
Describe the principles of protein sorting in terms of:
(i) Sorting signals
(ii) Folding status
(iii) Energy
(iv) Translocational Machinery
(i) Present in polypeptide primary sequence, direct cargo to cellular compartment (may be removed post-targeting)
(ii) Proteins may undergo folding/unfolding events during trafficking
(iii) Energy - required as compartmentalisation increases order/decreases entropy
(iv) Pores/Channels - to allow protein to cross membrane
What is the Nuclear Pore complex (NPC)?
What is its structure?
- Transport channel located in the nuclear membrane, which allows bi-directional transport of fully folded proteins in/out of nucleus
- Large Complex (125Mda) consisting of approx. 13 different protein species
Compare the Nucleocytoplasmic Transport of Large vs Small proteins?
(2 Points)
- Small proteins (~5kDa) - passively diffuse through NPCs
- Large Proteins (>30kDa) - require receptor-mediated transport through NPCs
What diseases are associated with NPC protein defects?
(1 Point)
ALS/MND
Define:
(i) Karyopherins
(ii) Types (Give Example)
(3 Points)
(i) Cargo receptors required for nucleocytoplasmic transport
(ii) Importins (e.g., Importin a/B) and Exportins (e.g., CRM1)
.Describe how cargo receptors transport proteins into/out of the nucleus
(2 Points)
- Receptor/Cargo complex undergoes series of weak/transient interactions between hydrophobic pockets of receptor and FG repeats in the NPC fibril proteins
- FG repeats form hydrogel meshwork that occludes pore, regulating transport
Describe the consensus of sorting signals for:
(i) Nuclear Import
(ii) Nuclear Export
(i) Nuclear Localisation Sequence (NLS) - consists of series of consecutive +vely charged residues (Arg, Lys)
(ii) Nuclear Exportation Sequence (NES) - consists of loose consensus of aliphatic residues (Val, Met, Leu)
What is the role of RanGTPases in Nucleocytoplasmic Transport?
How does it function as a molecular switch?
(3 Points)
- small monomer GTPase, who’s hydrolysis of GTP provides energy to displace loss of entropy
- Functions as molecular switch as:
1. Ran-GAPs in the cytosol activate its GTPase activity, causing hydrolysis of bound ATP -> ADP
2. Ran-GEFs in nucleus catalyse nucleotide exchange of GDP for GTP
In what state are most mitochondrial proteins translocated?
Translocated Post-translationally in a partially folded state (maintained by Hsc70)
Why do Mitochondrial Proteins often require multiple sorting signals?
Mitochondria consist of multiple subcompartments (e.g., OMM, IMM, IMS) and subsequent require rigorous sorting mechanics to ensure proteins arrive at correct compartment
What are the 3 Classes of Mitochondrial sorting signal
(4 Points)
- Presequences - present at N-terminus, and are often cleaved post-translocation
- e.g., Amphipathic Helix - with exposed face of +ve residues that is recognised by translocator proteins - Non-cleavable signals - typically present in hydrophobic proteins that are inserted into membrane
- Internal signals - typically within IMS proteins, often consisting of Cysteine rich regions
Describe the structure of TOM, including the role of each of the subunits
(4 Points)
Consists of:
* TOM40 - forms central pore
* TOM20/70 - peripheral receptors
* TOM22 - central receptor
* TOM5-7 - Assembly/Stability
How does TOM recognise its cargo?
(2 Points)
- Subunits 70/22 - recognise chaperones associated with protein via the co-chaperones
- Subunit 20 - recognises cargo via the amphipathic helix/ targeting sequence
How can OMM proteins be inserted into the membrane?
(2 Points)
- Protein sequence may contain stop transfer sequence, causing proteins release laterally into the membrane by TOM
- Protein (B-barrels Porins) may be transported into IMS, and associate with IMS chaperones before being passed to SAM for insertion into membrane
