Protein Localization
Peter Takizawa
Department of Cell Biology
•Organelles
•Distribution of organelles by microtubules
and motor proteins
•Signal sequences
•Examples of protein import
1. Role of signal sequences in targeting proteins.
2. Describe how proteins get into ER, mitochondria, nucleus and peroxisomes.
3. Why.
1. Illustrates important biochemical mechanisms that are common to many biological processes.
2. Classical work in cell biology.
3. Mutations in some pathways lead to disease.
Cells contain a variety of organelles that
perform specific and unique functions.
1.
2.
3.
4.
5.
6.
7.
Each organelle contains unique set of proteins and other components.
Organelles perform special and unique set of tasks.
Organelles increase efficiency by concentrating components of a common pathway in small environment.
Protect cell from harmful components.
Some organelles surrounded by membrane to prevent loss of material.
Should be familiar with function of each organelle.
Still image gives impression that organelles randomly distributed.
Cells use microtubules to position
organelles within their cytoplasm.
Golgi
Microtubules
Golgi fragments
1. Cells organize and localize organelles.
1.1. Position where needed.
1.2. Mitochondria at sites of greatest energy consumption.
2. Tether.
3. Transport through cytoplasm.
4. Microtubules play a critical role in organizing cytoplasm of cells.
5. Golgi localize to central region, near nucleus.
6. Disrupting microtubules leads to fragmentation of Golgi and distribution throughout cytoplasm.
Microtubules extend throughout the
cytoplasm.
1. Microtubules effective for organizing cell because they are long filaments that extend throughout cytoplasm.
2. Large enough to span most cells.
3. Allows cells to position organelles in specific regions.
Microtubules are a hollow tube of
tubulin dimers.
-tubulin
+
13 protofilaments
-tubulin
1. Heterodimer of alpha and beta tubulin.
2. Both bind GTP.
3. Assemble into filaments end on end to form protofilaments.
3.1. 13 protofilaments make a single microtubule.
3.2. Hollow lumen or center.
3.3. Extensive lateral contacts between subunits gives MT greater strength and allows it to grow longer than actin filaments.
3.4. Similar to pvc pipe.
4. Note that microtubules are polarized.
4.1. One end has alpha tubulin and the other end has beta tubulin.
4.2. Grow from their plus ends as dimers associate with plus end.
Microtubules grow from their plus ends.
Video shows growth of microtubules in vitro. Microtubules grow from their plus ends as more dimers are added to the filament. Occasionally, microtubule will
stop growing and then shrink as dimers fall off of plus end. Inside cells, the plus ends of microtubules are capped or stabilized so they don’t shrink. The
repeated growing and shrinking allow microtubules to explore different areas of the cell and cells can reposition or reorient microtubules. Note that the minus
ends of the microtubules emanate from a common center. This region is called the microtubule organizing center and stabilizes the minus ends of
microtubules.
Proteins link organelles to microtubule
network.
Microtubule organizing center (MTOC)
1. Cells stabilize minus ends through MTOC.
1.1. Contains large number of proteins -> centrioles.
1.2. Ring of gamma-tubulin that binds and stabilizes minus ends.
2.Location of MTOC determines orientation of MTs.
2.1. In center, MTs radiate outward -> plus ends toward plasma membrane.
2.2. Organelles contain proteins that bind MTs -> serve as tethers.
2.3. Orientation allows cells to control distribution of organelles.
2.4.
Kinesins and dynein mediate bidirectional
transport of organelles along microtubules.
Dynein
1.
2.
3.
2.
3.
4.
Motor proteins move along MTs carrying organelles.
Motor proteins move in either plus or minus direction.
Kinesins large family of motor proteins most of which move toward plus end.
Dynein move toward minus end.
Motors attach to organelles via receptors in organelle membrane.
By controlling binding and activity of motors, cell can dictate movement along MTs and position.
Cells regulate activity of motor proteins
to control distribution of organelles.
Organelles will often have both types of motors on their surface, allowing cells to adjust their position. For example, these melanocytes contain a pigmented
organelle called a melanosome. In some organisms, melanocytes position melanosomes in response to the amount of light. In the presence of light. kinesin
on the surface of melanosomes moves them to the periphery of cells. In the dark, dynein returns the melanosomes to the center of the cell.
Targeting proteins to specific organelles
Proteins synthesized in cytoplasm must get
to correct organelle.
1. Each organelle contains unique set of proteins and other components.
2. Organelles perform special and unique set of tasks.
3. How do cells get certain proteins to the different organelles?
3.1. What targets proteins to these organelles.
3.2. How do they cross the membranes surrounding the organelles?
Proteins are imported into organelles from cytosol
or transported between organelles.
Cytosol
Mitochondria
Peroxisomes
Nucleus
Endoplasmic Reticulum
Golgi
Late Endosome
Secretory Vesicles
Lysosome
Plasma Membrane
The synthesis of all proteins starts in the cytosol, but those proteins with signal sequences are delivered to their appropriate organelle. Many
organelles receive proteins through the secretory pathway. Proteins are initially translated and inserted into the ER. Proteins are transported to the
Golgi and then delivered to the different organelles. Some organelles receive proteins directly from the cytosol. The nucleus is a unique organelle
as it allows bidirectional movement of proteins.
Proteins contain signal sequences that determine
their final destination.
Function of Signal Sequence
Example of Signal Sequence
Import into nucleus
-Pro-Pro-Lys-Lys-Lys-Lys-Lys-Val-
Export from nucleus
-Leu-Ala-Leu-Lys-Leu-Ala-Gly-Leu-Asp-Ile
Import into mitochondria
Import into ER
Import into peroxisomes
+H N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Glu-Arg-Asn3
Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu
+H N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala3
Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln
-Ser-Lys-Leu-COO-
1. Proteins contain short sequences that guide them to correct organelle or intracellular destination -> signal sequence.
2. Signal sequence interacts with machinery on organelle that imports protein into organelle.
2.1. How does machinery get to organelle?
3. Signal sequences often found at N-terminus but can be located internally or at C-terminus.
4. Signal sequences often lack exact sequence conservation.
4.1. Can’t identify based on sequence.
4.2. Properties of amino acids that function as signal -> hydrophobic, charged.
5. Glycosylation can also act as targeting signal.
Small changes in signal sequences have profound
effects on location of proteins.
PEXN
Human
Nucleoside
Transporter
Mitochondria
PARS
Mouse
Nucleoside
Transporter
Plasma
Membrane
Slide illustrates how subtle changes in signal sequences can affect the localization of proteins. In humans, a certain nucleoside transporter contains a 4 amino
acid motif that targets it to mitochondria. In mice, the same protein contains two changes in this motif and this targets the protein to the plasma membrane.
If you experimentally change the motif in the human protein to the mouse version, the human transporter localizes to the plasma membrane. Is this medically
relevant?
Fialuridine is a uridine analog that is a potent
inhibitor of hepatitis B.
Human Mitochondrion
Nucleoside
Transporter
Mouse Mitochondrion
In the early 1990’s there was a clinical trial of a drug designed to treat hepatitis B in 15 patients. Hepatitis B is a virus that affects the liver, causing
inflammation. One common treatment for this type of virus is nucleoside analogs. Nucleoside analogs are similar to the nucleosides that are normally used to
synthesize DNA, so that they can incorporated into DNA, but they are altered so that they stop synthesis or affect the stability of the DNA. Fialuridine is a
nucleoside analog that was found to be effective in reducing hepatitis B in animal models. It was subsequently used in human trials and initially it appeared to
reduce the amount of virus. But then something went terribly wrong. Long after the clinical trials were concluded, researchers discovered something unique
about the way fialuridine affected human cells. Because humans have a certain nucleoside transporter that localizes to mitochondria, fialuridine can enter
mitochondria. Why is this bad? Fialuridine poisoned mitochondria, reducing their ability to generate ATP. Why wasn’t this discovered before the clinical trials?
Fialuridine was tested on animal models for safety, but those animals were mice and rats whose nucleoside transporter lacks the mitochondrial signal
sequence. Consequently, their mitochondria did not take up fialuridine and were not affected by the drug.
What happened in the clinical trials? By reducing the activity of mitochondria, fialuridine force energy-intensive cells to start using glycolysis to generate ATP.
This lead to an increase in lactate in the blood and a condition called lactic acidosis. Because the liver processes lactate, the increase production of lactate lead
to liver failure 7 patients,5 of whom died.
Protein import into the endoplasmic reticulum
ER signal sequences have a tripartite structure but
little sequence similarity.
1. Most proteins that enter ER contains signal sequence at N-terminus.
2. Signal sequence contains 3 domain.
2.1. Most important functionally is hydrophobic sequence.
2.1.1. Will interact with hydrophobic tails of lipids.
2.2. On N-terminal side is series of charge residues of varying length.
2.3. On C-terminal side is set of polar amino acids.
2.3.1. Sequence specifies cleavage by peptidase.
2.3.2. Removes signal sequence from protein.
3. Note lack of homology.
Most proteins imported co-translationally into the
ER.
Ribosome
mRNA
Translocator
Protein
1. Most proteins that enter ER do so co-translationally.
1.1. Inserted as they are made.
1.2. Must coordinate translation with insertion.
1.3. Recruit ribosomes to ER membrane.
1.4. Energy consumed during protein translation drives protein through membrane.
1.5. Protein passes across membrane unfolded -> smaller.
2. Co-translation prevents dangerous proteins from acting in cytosol.
2.1. Lysosomal proteins digest macromolecules.
2.2. Complete translation before insertion and protein may fold and become active.
3. How do cells ensure that ER proteins are translated on ER and not in cytoplasm.
3.1. Conformational change in regulating activity of proteins.
Signal recognition particle binds to ER signal
sequences and pauses translation.
Ribosome
mRNA
Protein
SRP
1. Coordination of translation and translocation are driven by SRP.
1.1. Complex of 6 proteins and one RNA.
1.2. SRP recognizes and binds to signal sequence.
1.2.1. Contains flexible, hydrophobic pocket.
1.2.2. Binds hydrophobic region in signal sequence.
1.2.3. Flexibility allows it to accommodate different hydrophobic sequences.
1.3. SRP wraps around ribosome and pauses translation elongation.
SRP receptor on ER binds SRP to recruit partially
translated protein to ER membrane.
SRP
Cytosol
Lumen
Translocator
SRP
receptor
1. SRP bound to ribosome and signal sequence interacts with receptor in ER membrane -> SRP receptor.
2. SRP receptor recruits nascent protein to ER membrane.
3. How does receptor differentiate free SRP from SRP bound to signal sequence and ribosome?
3.1. SRP undergoes conformational change when bound to ribosome.
3.2. Receptor likely recognizes conformation bound to ribosome.
Proteins threaded through translocator to cross the
ER membrane.
SRP
Cytosol
Lumen
Translocator
SRP
receptor
1. SRP receptor causes SRP to release from ribosome and signal sequence.
SRP receptor induces conformational change to weaken interaction.
2. Proteins cannot cross membrane due to size and charge.
3. Require protein channel -> translocator.
3.1. Transmembrane protein that forms aqueous pore.
3.2. Crystal structure shows several transmembrane domains that surround pore.
3.3. In absence of translation, pore contains helix that acts as plug, preventing small molecules from diffusing through pore.
4. SRP receptor brings ribosome to translocator.
5. Translocator contains several ribosome binding sites.
6. Translation continues through pore.
7. Signal sequence binds to site on pore to open -> start transfer sequence.
Chaperones in ER help proteins fold after
translocation.
Translocator
Newly made
protein
1.
2.
3.
4.
5.
6.
7.
8.
Proteins inserted into ER as in unfolded state.
Proteins must fold into correct conformation inside lumen.
At low concentrations, most proteins fold successfully on their own.
At high concentration, like in lumen where lots of proteins trying to fold, folding is less robust.
Proteins contain hydrophobic domains that cluster in center of protein
At low concentrations, these domains interact with each other.
At high concentrations, hydrophobic domains from neighboring proteins interact.
Chaperones such as BiP bind exposed hydrophobic domains in unfolded proteins and prevent aggregation with other proteins.
Integral membrane proteins contain stop
transfer sequence.
Stop transfer
C-terminus
Cytosol
Lumen
N-terminus
1.
2.
3.
4.
5.
Many proteins are integral membrane proteins.
These proteins contain another stretch of hydrophobic residues that function as stop transfer sequence.
Stop transfer sequence will function as transmembrane domain.
With this mechanism, N-terminus resides in lumen and C-terminus resides in cytosol.
If transported to plasma membrane, N-terminus outside cell.
5.1. Receptors.
6. Can you have opposite orientation?
Start and stop transfer sequences determine
membrane topology of proteins.
Stop transfer
Cytosol
Start
transfer
Lumen
1.
2.
3.
4.
5.
6.
7.
Many proteins span the membrane more than once.
Proteins contain series of start and stop transfer sequences that determine membrane topology.
For example, internal start transfer starts insertion with N-terminus facing outside.
Stop transfer sequence ceases translocation.
Translocator releases protein into membrane.
Both N and C-termini face cytosol.
Sequential start and stop transfer sequences
lead to multispan membrane proteins.
1. Proteins can span membrane several times.
2. Series of start and stop transfer generate proteins that crosses membrane several times.
3. Start and stop transfer sequences location dependent. Can switch function if positioned changed.
Protein import into peroxisomes
Peroxisomes detoxify chemicals, metabolize
fatty acids and synthesize key lipid.
Peroxisome
Microtubules
1. Several vital functions.
1.1. Metabolize many harmful chemical.
1.1.1. Phenols, formaldehyde, ethanol
1.1.2. Enzyme catalase oxidizes these substances.
1.1.3. Prominent in liver.
1.2. Metabolize fatty acids.
1.2.1. Oxidize carbon chains 2 at a time to form acetyl CoA -> important for metabolism.
1.3. Catalyze first step in formation of plasmalogens.
1.3.1. Lipids specifically found in myelin that wrap axons of neurons.
1.3.2. Increase conduction velocity of neurons.
Proteins inserted post-translationally into
PERSPECTIVES
peroxisomes.
Protein with
peroxisome signal
sequence
Cytosol
a Binding
PEX5
e Receptor recycling
b Transport
d Translocation
PEX4
c Docking
PEX14
PEX13
PEX8
PEX12
PEX10
PEX22
Peroxisome
Figure 1 | A general model of peroxisomal-matrix-enzyme import. The dynamic distribution of PEX5
indicates that matrix-enzyme import involves: a | binding of enzymes (red circles) by the import receptors
(shown
here
PEX5functioning
in green); b
transport of receptor–enzyme complexes to the peroxisome surface;
1. Protein import
is critical
foras
proper
of| peroxisomes.
2. Similar mechanism
to mitochondria.
c | docking
of receptor–enzyme complexes through protein–protein interactions with peroxisomal
2.1. Peroxisomal proteins contain signal sequence.
membrane
proteins,
as PEX14
(purple)
and, perhaps, PEX13 (grey); d | dissociation of
2.2. Sequence
binds receptor
thatsuch
targets
protein to
peroxisome.
receptor–enzyme
complexes
and enzyme
2.3. Protein
transits across membrane
through
channel. translocation through a proteinaceous pore (perhaps containing
2.3.1.
PassesPEX10
through
as PEX12,
folded protein.
PEX8,
and
all shown in blue); and e | receptor recycling, which might involve PEX4 and
2.4. Proteins involved in protein import called Pex.
PEX22 (both shown in orange). PEX, peroxin.
nsPME–P
synthesis
to these p
as ‘preimp
We see
process tha
centration
cytoplasm
(FIG. 2). In s
for nsPME
example, p
ize rapidly
are more
enter pre
oligomeri
tively high
preimplex
proteins sh
they conta
preimplex
must oligo
plex forma
interaction
lent. Howe
centration
and affini
PEX5) ma
proportio
oligomeriz
mation in
Mutations in Pex proteins lead to loss of
peroxisomes.
pex1
Normal
on 13
patients.
ce chroof exon
from get 4756, a
xon 13
ubcloned
suballele.
ence ene Cells
(WT)that contain mutations in the Pex genes can’t import porteins into peroxisomes. Consequently, the protein content of peroxisomes can’t be
maintained and the cell loses its peroxisomes.
13insT)
is numf the murlined to
of the
xon 13
ermina-
Pex mutations cause Zelleweger Syndrome and
demyelination of nerves.
1. Several diseases associated with defects in genes that encode peroxisomal proteins.
2. Set of diseases that result from mutations in genes that encode proteins involved in import into peroxisomes.
2.1. These mutations result in an absence of peroxisomes at birth.
2.2. Results in build up of toxic materials including copper and iron-> enlarged liver.
2.3. Lack myelination around axons.
2.3.1. Loss of muscle tone, inability to move and suckle.
2.3.2. Can’t synthesize lipid for myelin.
3. Poor prognosis.
3.1. Death by 6 months.