Lecture 9 and 10 Flashcards
What is protein sorting?
It is the movement of proteins within cell from different compartment, into the cell of out of the cell
Where is protein synthesis initiated?
Protein synthesis is initiated on ribosomes in sytosol
Where do UNFOLDED proteins that were synthesized in the cytosol go
mitochondria and plastids
Where do FOLDED proteins that were synthesized in the cytosol go
nucleus and peroxisomes
Co-translational process of protein sorting and synthesis
They are proteins with an ER signal sequence, they are associated with the ER during protein synthesis
Where does gated transport occur
proteins moving between cytosol and nucleus
Transport of cargo through nuclear pore complex
There is transport in both directions
1. nuclear import and nuclear export
Nuclear Pore Complex
- There is selective transport of macromolecules
- Free diffusion of small molecules (<5000 daltons)
What is the nuclear pore complex made of?
Nucleoporins q
What is the nuclear localization signal rich in?
Lysine and arginine
What does the nuclear import receptor bind to and where does it transport into?
The nuclear import receptor binds to a nuclear localization signal and nucleoporins in the nuclear pore complex. it transports into the nucleus.
What does nuclear export receptor bind to and transport into?
The nuclear export receptor binds to the nuclear export signal (NES) and to nucleoporins in NPC. It transports into the cytosol.
What does Ran GTPase cyle between?
GDP bound (to nucleus by Nuclear transport factor 2) and GTP bound (to cytosol with nuclear import and export receptors).
What is RAN GTPase regulated by?
Ran-GAP (GTPase activating protein) - stimulates GTP hydrolysis by Ran
Ran-GEF (Guanine nucleotide Exchange Factor) - promotes the exchange of GDP for GTP by Ran
Where is Ran-GAP found?
Cytosol
Where is Ran-GEF found?
Nucleus
Where are high and low Ran-GTP found?
high Ran-GTP - nucleus
low Ran-GTP - cytosol
Nuclear import of cargo proteins
- Nuclear import receptor binds cargo in the cytosol.
- Receptor and cargo move to the nucleus.
- Ran-GTP binding: causes cargo release
- Empty import receptor and Ran-GTP move to the cytosol
- Ran Binding protein and Ran GAP promote GTP hydrolysis and release of import receptor
Nuclear export of cargo proteins
- Nuclear export receptor binds Ran-GTP and cargo in the nucleus
- Receptor, cargo and Ran GTP move to cytosol
- Ram Binding Portein and Ran GAP promote GTP hydrolysis, release of cargo and release of export receptor.
- Empty export receptor returns to the nucleus.
Nuclear Factor of activated T-cells
(see diagram from notes)
High intracellular Ca2+ - nuclear import
Low intercellular Ca2+ - nuclear export
Experiment with NFAT
- Express NFAT-GFP fusion protein in T cells
- Add calcium ionophore
- Monitor fluorescence
Transmembrane transport
ER, mitochondria, plastids, peroxisomes
Protein translocators
- transport of protein across membrane
- protein usually unfolded
Protein translocators in mitochondrial matrix
TOM and TIM23 complexes
Sorting proteins to mitochondria and chloroplasts
they have their own genme and ribosomes but most proteins are nuclear encoded
1. they are translated in cytosol
2. imported into organelle - post translational, proteins remain unfolded by association with hsp70 chaperones
Importing proteins to the mitochondrial matrix
Precursor protein has a mitochondrial signal sequence (peptide) N terminal amphipathic alpha helix
it binds the receptor and moves through TOM and TIM23 complexes into matrix space signal sequence cleaved
Importing proteins to the chloroplast
They have TOC and TIC translocators
the precursor protein has a chloroplast signal sequence - N terminal amphipathic alpha helix
Signal sequence cleaved in chloroplast
Different from mitochondrial signal sequence for correct targeting in plants
If targeting to thylakoid
1. hydrophobic thylakoid signal sequence
2. unmasked when chloroplast signal sequence cleaved
Sorting proteins to peroxisome
precursor protein
1. peroxisomal targeting signal
2. 3 amino acids at C terminus (SKL)
3. protein folded
it is transported across the membrane by a large translocator complex
How can proteins be further sorted to other compartments or to cell surface from ER?
Vesicular transport
2 major functions of the ER
Synthesis and modification of proteins
Synthesis of lipids
What do proteins sorted to the ER have?
ER signal sequence
The proteins include soluble proteins, transmembrane proteins, proteins destined for golgi, secretion, lysosomes
Protein sorting to ER
- mRNA + ribosomes
- translation starts, the ER signal sequences (hydrophobic amino acids at N terminus) emerges first
- The ribosomes are directed to ER membrane
- Co-translational translocation takes place
What do signal recognition particles and signal recognition receptors have?
They have GTPase domains that bind GTP
SRP + ribosome
low affinity
SRP + ribosome + ER signal sequence
high affinity and binds SRP receptor
What does ribosome form a tight seal with?
it forms a tight seal with the translocator to prevent diffusion of ions and small molecules
What happens when only SRP and SRP receptor are bound?
There is GTP hydrolysis and the complex dissociates, and SRP is released.
here translation continues and translocation begins.
What causes translation to slow down?
Binding of SRP to ribosome
What is the ER signal sequence ?
N terminal start transfer sequence - hydrophobic amino acids
What cleaves ER signal sequence?
Signal peptidase
Where happens to both ER signal sequence and protein?
The ER signal sequence laterally diffuses into the lipid bilayer (translocator is gated in a 2nd direction) and translocated protein is released into ER
How many types of insertions for single-pass transmembrane proteins?
3
Single pass (1)
- The ER signal sequence is an (NH2) start transfer sequence.
- The transmembrane domain is a stop transfer signal that laterally diffuses into liid bilayer.
- signal peptidase cleaves start transfer.
protein synthesis continues in cytosol with COOH end in cytosol.
Single pass (2 and 3)
- Transmembrane domain: internal start transfer sequence that is not cleaved.
- this internal start transfer sequence will laterally diffuse into the lipid bilayer.
Orientation in single pass (2 and 3)
The more positive side is the cytosolic side. It is determined by amino acids flanking the internal start transfer sequence.
Example 1 of multipass transmembrane protein
1st transmembrane domain: internal start transfer sequence
2nd transmembrane domain: stop transfer sequence
Both N and C terminus face cytosol
Example 2 of multipass proteins
RHodopsin
1st transmembrane domain: start transfer; (+) amino acids
2nd transmembrane domain: start transfer
3rd: stop 4th: start
Membrane protein types
integral, lipid anchored and pripheral
where are most transmembrane proteins glycosylated?
extracellulcar face
Formation of GPI-anchored proteins
- Target protein has C terminal hydrophobic domain.
- GP anchor is preformed in the membrane.
- The transmembrane domain is signal for the GPI anchor.
- Er enzyme transfers protein to the GPI anchor.
- GPI anchored protein ends up on ER luminal side and can go to cell exterior surface.
What serves as a signal for the gpi anchor?
C terminal hydrophobic domain
