BIOM20001 MCB 137 Protein traffic in the cell
We struggle with:
• ACCESS
o Getting from the cytosol into various compartments o Due to topology
o 3 mechanisms involved for getting access
• TARGETING
o Getting to the right compartment 3 Mechanisms for Access
1. GATED TRANSPORT (red)
• Movement between cytosol and nucleus
• Topologically equivalent
2. TRANSMEMBRANE TRANSPORT (blue)
• Movement to lumen of mitochondria, plastids, peroxisomes and ER
• NOT topologically equivalent
• Involve protein translocators 3. VESICULAR TRANSPORT (green)
• Movement between ER and other organelles plus cell surface
• Topologically equivalent
• Getting to the right compartment involves correct targeting Mini-questions
1. Many cell surface transmembrane proteins, called glycoproteins, have an attached carbohydrate group, which faces the extracellular space. This carbohydrate group is attached to the protein when it is still in the rER. Is the carbohydrate attached to the side of the protein that faces the lumen of the rER or the cytosol?
The carbohydrate would be attached to the side of the protein that faces the LUMEN of the rough endoplasmic reticulum because the extracellular space is topologically equivalent to the lumen of organelles, such as the rER, therefore if the carbohydrate faces the extracellular space after transportation, it uses vesicular transport.
2. Explain why the orientation of a transmembrane protein (i.e. which side of the protein faces the inside and which side faces the outside of the membrane) does not change when it moves from the rER to the Golgi apparatus to the plasma membrane.
The fusing and budding of vesicles ensures that the inner and outer membranes always faces particular conditions, such that the outer membrane of the ER will be the outer membrane of the Golgi.
3. Topology is commonly misunderstood by students. What is meant by ‘two cellular compartments being topologically identical’?
Means that molecules that are inside the organelles vesicles can move into another through vesicle fusion without touching the membrane. Occurs when each compartment’s interior environment is identical to the other.
SAMPLE
BIOM20001 MCB 139 Nuclear import receptors
• Cargo proteins destined for nucleus bind to specific Nuclear import receptors via their Nuclear Localisation Signals
• Nuclear import receptors interact with Nuclear Pore Complex proteins to transfer cargo in/out of nucleus
• Binding and dissociation of nuclear import receptors to nuclear pore complex proteins transports CARGO INTO NUCLEUS
1. Cargo with NLS binds NIR
2. NIR shuttles into the nucleus via the NPC (F-G repeats) 3. Ran GTP binds to NIR to discharge the cargo.
4. NIR shuttles out of the nucleus via the NPC (F-G repeats) 5. Ran-GTP is hydrolysed to Ran-GDP in the cytosol
6. NIR is free to shuttle more cargo into the nucleus
• Nuclear import receptors cycle between cytosol and nucleus
• Similarly, the EXPORT of proteins and RNA from nucleus works in the same way but REVERSE
1. Cargo with NLS binds to NIR
2. Ran-GTP also binds to NIR in the nucleus 3. NIR shuttles out of the nucleus via the NPC
4. Ran-GTP is hydrolysed to Ran-GDP in the cytosol and releases the cargo
5. NIR is free to shuttle cargo in/out Compartmentalisation of Ran-GTP and Ran-GDP
• Ran is a small GTPase
• Differential Ran activity by compartmentalisation of regulators
• Confers directionality on transport
• Ran-GDP phosphorylated in the nucleus and Ran-GTP is hydrolysed in the cytosol by different regulators
Regulation of Nuclear protein imports
• NFAT = transcription factor, found in the cytosol
• NLS in NFAT is cryptic but exposed after dephosphorisation of amino acid residues (Ser) by a Ca2+
regulated kinase (calcineurin)
• Dephosphorisation produces a conformational change
o Now has a nuclear localisation sequence to enter the nucleus
• Fate decisions in embryos determined by nuclear accumulation of specific transcription factors o Critical for formation of embryo
• Dorsal protein
o Determines ventral axis because it is only expressed ther
o Nuclear localization only in ventral cells of early Drosophila embryo
• Mutation of Dorsal results in dorsalization of embryos (no ventral structures)
SAMPLE
BIOM20001 MCB 140
TRANSMEMBRANE TRANSPORT
Recall that this is the second main way of accessing compartments within a cell. Transmembrane transport involves movement into the mitochondria and plastids; lumen of peroxisomes and endoplasmic
reticulum. Involves protein translocators.
Transport of proteins into the MITOCHONDRIA
• Mitochondria have 2 membranes that need to be crossed for proteins to be targeted correctly
• Mitochondrial proteins
o Imported as fully synthesised but unfolded polypeptide chains o Have a specific targeting/signal sequence
o Do not fold, chaperones prevent folding
• Translocation depends on amphiphilic signal sequences o Very specific
o Forms an ⍺-helix à non-polar and polar (+ve charged) residues on separate sides of helix
o Hydrophobic region matches hydrophobic groove of receptor
• Protein translocators
o Multiprotein complexes embedded in outer and inner membranes
o Move mitochondrial proteins through both membranes o Act as receptors for cargo proteins, translocation channels
§ TOM complex – Transport/insert proteins through outer membrane
§ TIM complexes – Insert/transport proteins through inner membrane (OXA into matrix)
o Protein “snakes” through translocators in unfolded state 1. Signal sequence binds to TOM complex
2. TIM complex aligns with TOM complex
3. Protein is translocated through both TOM/TIM complexes
4. In the matrix, signal sequence is cleaved 5. Protein is able to fold into a mature mtProtein o Import requires energy (ATP)
§ Chaperone proteins (cytoplasmic Hsp70) bind to precursor peptide
§ Release requires ATP hydrolysis to ‘push’
protein through TOM complex
§ Import via TIM results in mitochondrial Hsp70 binding
§ ATP hydrolysis to ‘pull’ protein through TIM complex