Intracellular compartments and protein sorting Flashcards
1
Q
A
2
Q
Why do cells need sorting?
A
- A bacterial cell has no compartments,
yet functions well. - Membrane compartments in
complex eukaryotic cells provide
different local environments that
facilitate specific metabolic
functions (incompatible processes
can go on simultaneously inside the
same cell) - Organelle characteristics: specific
lipid & protein composition,
metabolism, structure, abundance,
arrangement - An animal cell contains 10 billion
protein molecules of 10,000 different
kinds - Almost all synthesised in cytoplasm,
need to find their way to the correct
location to be functional
3
Q
Describe phospholipid bilayers and compartment formation
A
- All lipid molecules in the cell are
amphiphilic (polar+nonpolar) - Phosphoglycerides have two
associated long-chain fatty acids, one
of which is unsaturated (C=C kink) - Different head groups provide
different properties - Cholesterol molecules are able to sit
in the gap caused by the kinks. - The steroid rings partly immobilise
the hydrocarbon chains, decreasing
mobility and permeability of the
membrane - The presence of two (rather than
one) fatty acid chains mean the
phospholipids will form bilayers, not
micelles - Sheets of bilayers will form enclosed
compartments, to avoid exposure of
hydrocarbon tails to water - Individual lipid molecules rapidly
diffuse laterally at around 2mm/sec - Phospholipid translocators ensure
flip-flop across sides of the bilayer - Addition of cholesterol to a mix of
phospholipids causes formation of
“rafts”
4
Q
How to move proteins between compartments?
A
- Cells use three different ways:
– Gated transport (selective gates that
actively transport macromolecular
structures, while allowing diffusion of
small molecules)
– Transmembrane transport (protein
translocators directly unwind and
pull specific proteins from cytosol to
a topologically different space)
– Vesicular transport (membrane
enclosed vesicles get loaded with a
cargo, physically move, and offload
cargo via membrane fusion to a new
compartment
5
Q
Describe sorting signals
A
- Most sorting signals are encoded by the amino acids at the end of a polypeptide
- Signal peptidases remove the signal after transport
- Some signals are spaced out along the protein, coming together in 3D to form a “signal patch”
Recognition based on protein-protein interactions forms the basis of most cellular
processes
6
Q
How do we use molecular biology to study the mechanism of protein translocation?
A
- Identify the minimal sequences that
are responsible for targeting a
particular protein to a particular
compartment - Take that sequence and fuse it to a
reporter gene (e.g. GFP) - Express it in cells
- Use site-directed mutagenesis to
alter single amino acids within the
sequence, to determine which
structural elements are important
7
Q
Describe nuclear transport in terms of the NPC
A
Nuclear pores
* Built of 5 types of units, consisting in
total of only 30 proteins:
– Annular subunits (central)
– Lumenal subunits (TM)
– Ring subunits (faces)
– Fibrils (FG repeat proteins)
– Nuclear basket
* Unstructured regions of proteins in
the central pore form a tangled
network, restricting movement
* Molecules < 5kDa move freely
through
* Macromolecules > 40kDa need to be
actively transported
8
Q
Describe the nucleolus
A
- largest structure in the nucleus of eukaryotic cells
- site of ribosome biogenesis
- participates in the formation of signal recognition
particles
9
Q
How does nuclear transport work?
A
- Nuclear Localisation Signals (NLSs)
can be located anywhere in the
protein - Are +vely charged
- Only one protein in a complex need
have one for all subunits to be
transported - Recognised by nuclear import
receptors (directly (A), or indirectly
(B)) – also called karyopherins, or
importins - Different members function to
export, as well as import
10
Q
What is the importance of Ran?
A
- Ran is a small GTPase – a molecular
switch - It is found in two states – bound to GTP
(active) or bound to GDP (inactive) - Is switched off by GTPase Activating
Proteins (GAPs), which increase the rate
of GTP hydrolysis - Is activated by Guanine nucleotide
Exchange Factors (GEFs), which increase
the rate of exchange of bound GDP for
GTP - The GAP is present in the cytosol – so
Ran.GDP predominates here - The GEF is present in the nucleus – so
Ran.GTP predominates here
11
Q
Describe the role of Ran in nuclear import
A
- Karyopherins do not bind Ran.GDP.
Therefore, Karyopherins that are in the
cytosol are free to bind cargoes (for
import) - When Karyopherin-cargo makes it across
the NPC, they encounter Ran.GTP, which
binds the karyopherin, replacing the
cargo - The Ran.GTP:Karyopherin gets
transported back to the cytosol, where
GTP hydrolysis is stimulated by the GAP,
releasing free Karyopherin - Ran.GDP gets transported back into the
nucleus via its own nuclear transport
receptor, and is converted back to
Ran.GTP by the GEF - Nuclear export occurs when specific
proteins interact with Karyopherins in a
Ran.GTP specific manner
12
Q
Describe entry of transport into mitochondria
A
- Mitochondria are double membranebound, with two active spaces:
– Matrix
– Inter-membrane space - Proteins to be imported are fully
synthesised on cytoplasmic
ribosomes, but not folded - Held as polypeptides by Chaperones
and other proteins - Signal sequences determining entry
through to the matrix form
amphiphilic a-helices with +ve
charged clusters on one side - These bind to protein translocator
complexes (TOM, TIM)
13
Q
Describe transport into the mitochondria
A
- TOM complex binds the signal sequence to facilitate transport across outer membrane with ATP
hydrolysis causing concomitant dissociation of Chaperones (Hsp70 family) - Sequence further into the protein then binds the TIM complex. Mitochondrial membrane
potential drives initial translocation of +vely charged residues through to the matrix - Immediately bound by a mitochondrial Hsp70
- ATP hydrolysis releases the Hsp70 from the polypeptide and drives completion of import
- Mitochondrial Hsp60 (not shown) then folds the proteins correctly
14
Q
Describe the different end points of mitochondrial transport
A
- Inner membrane proteins either:
– (i) have a hydrophobic region following the +vely charged residues that stop TIM23 from
translocating the protein through
– (ii) have a second signal sequence facilitating transport back from the matrix to intermembrane space via the OXA translocator (same one used for mitochondrial-encoded
proteins)
– (iii) metabolite transporters are pulled through TOM as a loop, and bound by intermembrane chaperones. Guided to a second TIM complex (TIM22) which inserts them into
the membrane - Inter-membrane space proteins follow route (i) or (ii) (if not synthesized in the
mitochondria), but the second hydrophobic signal is cleaved by a specific signal
peptidase
15
Q
How can we use biochemistry to study the mechanism of protein translocation?
A
- A radioactively labelled, in vitro
translated protein is incubated
with and without purified
organelles - Can test for translocation via:
– (i) co-fractionation during
centrifugation
– (ii) reduction of size of protein (due
to protease cleavage)
– (iii) incubation with exogenous
proteases (protected if in the
organelle)
16
Q
Describe transport into chloroplasts
A
- Similar principles to mitochondrial
transport, but different topological
problems (the thylakoid space
requires an additional transport step) - A second hydrophobic sequence is
unmasked after the first is cleaved in
the stroma - A number of different proteins
facilitate thylakoid transport - Chloroplasts use energy from GTP
and ATP to get photosystem proteins
across the double membrane, then
the H+ gradient across the thylakoid
membrane
17
Q
Describe using genetics to study the mechanism of protein translocation
A
- Most experiments carried out in
yeast as a model organism - Endogenous Histidinol
dehydrogenase (which produces
Histidine) is replaced with HisDH
fused with an ER targeting sequence - Cells die in media lacking His, as the
HisDH is sequestered in the ER - BUT, if a random mutagenesis screen
knocks out a gene required for ER
transport, cells will live - Also used to produce temperature
sensitive mutations
18
Q
Describe peroxisomes
A
- ubiquitous organelles
- single membrane (0.1-0.5 µm, no DNA)
- essential for human health and development (lipid metabolism, e.g. myelin lipids,
H2O2/ROS metabolism) - dynamic organelles, high plasticity („multipurpose“ organelles)
- responsive to environmental stimuli
- interesting biology (e.g. import of folded, cofactor-bound, and oligomeric
proteins; de novo formation; signalling platforms; ageing; neurodegeneration)
19
Q
Describe the biogenesis of peroxisomes
A
Peroxisomal Targeting-Signals (PTS)
* C-terminal tri-peptide (PTS1) [SKL]
* N-terminal nona-peptide (PTS2) [(R/K)(L/V/I)X5
(Q/H)(L/A)]
PTS1 and PTS2 are recognized by specific receptors in the cytosol
[Pex5 for PTS1, and Pex7 for PTS2]
20
Q
Peroxisomal matrix protein import
A
unique cellular process, soluble receptors,
import of folded and oligomeric proteins, transient pore (?)
21
Q
Describe the transient pore model
A
- Protein import is ATP
independent - only recycling of Pex5 requires
ATP - PTS1‐containing cargo recognised by
peroxisomal import receptor Pex5 - Multiple Pex5 subunits may integrate into a
larger complex in the peroxisomal membrane - Other proteins (Pex14, Pex13, Pex17) aid
tethering the receptor to the membrane and in the
assembly, stabilization and rearrangement of
the translocation apparatus. - Cargo release then initiated by
intra‐peroxisomal factors (e.g. Pex8) - Pex5 disassembly and recycling triggered by
Pex1 and Pex6 ATPases. - Mono‐ or di‐ubiquitylation are reversible steps
for efficient recycling of import receptors - Polyubiquitylation leads to proteasome
dependent degradation of receptors when the
physiological dislocation of receptors is blocked.
22
Q
Describe transport into the ER
A
- Difference in density of smooth ER
and rough ER microsomes allowed
their purification for use in in vitro
studies, and pioneered the signal
hypothesis - Proteins are transported into the ER,
co-translationally (almost always) - The ER captures two types of
polypeptides:
– (i) transmembrane proteins
– (ii) water soluble proteins for
secretion or other organelles (Golgi,
lysosomes)
23
Q
Desribe the signal recognition particle
A
- Binds the N-terminal signal peptide
- Consists of 6 proteins + an RNA molecule
- Has a large hydrophobic pocket lined by
methionines (unbranched and flexible to
accommodate lots of different
combinations of non-polar amino acids) - It wraps around the large ribosome
subunit, capturing and protecting the
polypeptide as it emerges, and blocking
further translation - Binding causes a conformational change
such that the SRP can bind an SRP
receptor in the ER membrane - This triggers interactions with a protein
translocator
24
Q
Describe the mechanism of ER transport
A
- The Translocator is a conserved
hetero-trimeric complex collectively
called Sec61 - Composed of a-helices which bundle
around each other, leaving a central
pore - The pore is normally closed by a
small a-helix (which will stop
molecules like Ca2+ ions from freely
moving between compartments) - The signal peptide can displace the
short helix, allowing the polypeptide
to be transported
25
Q
Describe the differnt types of proteins transported
A
- Soluble proteins:
– On binding an ER signal sequence, the polypeptide chain is completely transferred
into the ER
– Signal peptidase then cleaves the signal sequence - Single-pass TM proteins (I):
– The polypeptide contains a stop transfer signal (hydrophobic sequence)
– Signal peptidase cleaves the signal sequence
– The C-terminus remains in the cytosol - Single-pass TM proteins (II):
– If a polypeptide has an internal signal sequence, it still gets directed to the
translocator
– If there are more +vely charged residues before the hydrophobic core of the signal
sequence, the N-terminus is inhibited in translocation, resulting in complete transfer
of the C-terminal part of the polypeptide
– The reverse is true if there are more +vely charged residues after the internal signal
sequence (i.e. the N-terminus is pulled through and the C-terminus remains cytosolic) - Multi-pass TM proteins:
– Internal signal sequences serve as
start-transfer signals
– Continues until a stop-transfer signal
is encountered
– For double-pass membrane proteins,
this is enough
– For more complex proteins, a second
start-transfer sequence re-initiates
translocation
