lecture 7 Flashcards
Protein trafficking
1
Q
What sorts of compartments are there within a cell?
A
- nucleus surrounded by nuclear membrane
- continuous with endoplasmic reticulum
- golgi apparatus
- lysosome
- endosome
- peroxisome
- mitochondria
2
Q
What are the topological relationships between compartments in a eukaryotic cell?
A
- the plasma membrane separates the inside of the cell from the outside
- but not quite this simple
- the inside of the organelles is more similar to the outside of the cell
- not completely true that e.g. the inside of the golgi apparatus is exactly the same as outside the cell but whatever was on the outer surface of the plasma membrane will be on the inner surface of the organelles
3
Q
What are the three basic types of protein trafficking?
A
- gated transport: in and out of the nucleus
- transmembrane transport: transports proteins into peroxisomes, mitochondria, plastids, and the ER
- vesicular transport: transport between vesicles (ER, Golgi, secretory, late endosome, lysosome, early endosome, cell exterior)
- the first forms of transport are very individual while vesicular transport might involve the movement of many hundreds/thousands of proteins at once
4
Q
What directs proteins to their correct ‘address’ in the cell?
A
- signal sequences are like Post codes for proteins
e.g. import into nucleus= pro-pro-lys-lys-lys-arg-lys-val
while export from nucleus = leu- ala-leu-lys-leu-ala-gly-leu-asp-ile
5
Q
Describe the nuclear envelope
A
- the nucleus is surrounded by a double-membrane envelope that is penetrated by nuclear pores and is continuous with the endoplasmic reticulum
6
Q
What is the nuclear pore complex?
A
- the nuclear pore is not just a simple hole
- an intricate structure of various proteins and fibrils
- like a basketball hoop
- fibrils important for recognising proteins that enter the nucleus
- it is a gated diffusion barrier
- the limit size for free diffusion is around 60 kD (amino acids, ions, small organic molecules can freely diffuse through this pore)
- larger molecules can be actively transported through the NPC
7
Q
What are the nuclear import receptors?
A
- proteins that need to go into the nucleus (cargo proteins) bind to specific nuclear import receptors via nuclear localisation signals (NLS)
- nuclear import receptors interact with NPC proteins to transfer cargo in/out of the nucleus
8
Q
What gives directionality to nuclear transport?
A
Ran GTPase
- functions like a molecular switch
- ON in the nucleus, OFF in the cytosol
- Ran-GAP is located in the cytosol - assists the Ran-GTPase to hydrolyze the GTP back to GDP
- Ran-GEF is located in the nucleus - guanine exchange factor, assist in the exchange from GDP to GTP
9
Q
Describe the model of nuclear transport.
A
Import:
- Cargo with NLS bind NIR
- NIR shuttles into the nucleus via the NPC
- Ran GTP binds to NIR to discharge the cargo
- NIR shuttles out of the nucleus via the NPC
- Ran-GAP activates Ran-GTP hydrolysis to form Ran-GDP in the cytosol
- Ran-GDO actually has its own NIR to shuttle it back into the nucleus where it is converted to Ran-GTP by Ran-GEF
Export:
- Export of Cargo from the nucleus is similar except that:
- Ran-GTP promotes binding of cargo (with nuclear export signal) to Nuclear Export Receptor (NER/exportin)
- Ran-GDP dissociates cargo from the NER
For both export and import, cycling of the NIR and NER through the pore is independent of Ran-GTP and cargo
Ran-GTP provides directionality.
10
Q
What is the importance of the NLS?
A
Nuclear localisation requires an intact and functional NLS:
- normal Lys-rich sequence targets a protein to nucleus
- mutation of Lys->Thr causes cytoplasmic retention
- Add an NLS to a cytoplasmic protein -> nuclear localisation
11
Q
What are the particular mechanisms/complexes of different organelles in regards to transmembrane transport of proteins?
A
- mitochondria: TOM/TIM complexes
- chloroplasts - SRP-like, Translocators
- peroxisomes - Peroxins
- Endoplasmic reticulum: Ribosome, SRP/Sec61 translocators
- all of these require energy: usually ATP/GTP hydrolysis
12
Q
What does transmembrane transport mean?
A
- transport of a protein ACROSS a membrane
- NOT transport of ‘transmembrane proteins’
13
Q
How does transmembrane transport occur across a mitochondria?
A
- Cytosolic protein associated with small Hsp70 proteins keeping it in an unfolded state
- Binds to a receptor on the outer mitochondrial membrane that is part of the TOM complex
- With the addition of energy the Hsp70 proteins dissociate and the protein is fed through a translocator in the TOM complex
- then passess through the TIM complex on the inner mitochondrial membrane (this also requires energy)
14
Q
How does transmembrane transport occur across the endoplasmic reticulum?
A
- the act of getting proteins into the ER couples translation with their transport
- in the cytosol there are free ribosomes/polyribosomes which will translate proteins
- proteins that are being transported into the ER end up being translated by ribosomes bound to the ER
- before it is even its fully folded state it is fed through the translocator into the ER, where it then completes folding
- transport into the ER is the first step in getting proteins into other organelles - from here it goes via vesicular transport
15
Q
How do ER signal peptides and SRP direct ribosomes to the ER membrane?
A
- peptide synthesis is initiated on FREE ribosomes
- signal sequence of nascent polypeptide emerges from ribosome, binds an SRP (Signal Recognition Particle) and pauses translation
- SRP binds SRP receptor and directs ribosomes to the ER membrane
- Ribosome pore docks with translocator and SRP released
- Translation contintues into the ER lumen
