- We’re going to look at sorting of proteins into different intracellular
- We’ve been looking at the movement of small molecules across the different
membranes in a cell but the proteins also need to move to the different
compartments in order for the compartments to have their specific functions.
For example, the ER, the Golgi, the peroxisome and the nucleus all need
specific proteins in order to maintain their proper functions. For example you
can imagine that a transcription factor needs to get to the nucleus in order to
transcribe DNA into RNA so that transcription factor needs to be sorted to the
nucleus in order to function properly.
- Again the 1 lecture we mainly went over membrane components & looked at
the transport of small molecules across these membranes & now we’re going to
look at the sorting of proteins across these membranes into the different
compartments within a cell. For example, the mitochondria & we’re going to
look at cytosolic proteins, the peroxisomes, the ER, & of course the nucleus.
We’re also going to look at the nucleus & how proteins also get into & out of
- For today, we’re going to look at protein sorting into the nucleus,
mitochondria and into the peroxisomes.
Within cell from different compartments
Out of cell
- Now we can look at the movement of proteins. Don’t forget that the
membrane surrounds the cell itself, the PM and also the organelles within the
cell. The peroxisomes are surrounded by a membrane, the nucleus is actually
surrounded by two lipid bilayers, mitochondria also have a lipid bilayer
surrounding them, the Golgi, the endoplasmic reticulum, all of these are
compartments which contains proteins and need proteins sorted to them.
- We can look at movement of proteins within the cell from different
compartments, we’re also going to look at the movement of proteins out of the
cell across the plasma membrane and then how do cells uptake proteins from
the extracellular environment in a process termed endocytosis.
- So how do proteins get from outside the PM to the inside of the cell? There
are thousands of proteins synthesized in a cell and the protein synthesis is all
initiated on ribosomes in the cytosol. Initiation if you’ll recall from BIO240 in
the fall, you will recall that DNA is transcribed into RNA, the RNA is shuttled
into the cytoplasm where it is translated by ribosomes into proteins. All
proteins, the initiation of translation are all initiated in the cytosol. - From the cytosol, proteins need to be sorted to their proper locations. An ER
protein needs to be translocated from the cytosol to the ER and to do this, the
proteins have certain signaling sequences on them that guide them to the proper
locations. The amino acid sequence contains a peptide or a signature sequence
that will guide the proteins to their appropriate compartments.
- So just appreciate the thought that protein synthesis of almost all proteins is
initiated in the cytosol.
Proteins fully synthesized in cytosol before sorting
Associated with ER during protein synthesis
- The different types of protein sorting can occur post-translationally, so in a
post-translational process, the proteins are fully synthesized in the cytosol
before being sorted. The entire protein is made and then it is sorted. This
occurs for sorting to the mitochondria, the plasmids or chloroplasts, nucleus
and to the peroxisomes.
- There are two different types. In one type, the protein remains unfolded.
There are usually specific proteins called chaperones that maintain these
proteins in the unfolded state before they’re sorted to the mitochondria or to the
chloroplast. In the second type, the proteins are completely folded after their
translation and then the folded protein is translocated into the nucleus or to the
peroxisomes. In both cases, these occur post-translationally.
- In contrast, there is also co-translational sorting that occurs and this occurs
into the endoplasmic reticulum. So proteins with an ER signal sequence will
associate with the endoplasmic reticulum during protein synthesis.
- We’re going to go into details about this in the next lecture but basically, this
just shows us what this kind of co-translational process looks like, here would
be the ribosome, the mRNA is being translated into protein and as the protein is
being translated, the protein is being funneled into the ER across the ER
membrane. This is sorting in a co-translational manner. As the protein is being
translated, it is being sorted to the ER, you can see that the ribosome is closely
associated with the ER and that is what gives the appearance of the rough ER,
is the setting of ribosomes all around the ER.
- There are three main types of transport that we’re going to cover.
- Gated transport which refers to the movement of proteins from the cytosol to
- Transmembrane transport which occurs to the endoplasmic reticulum as we
just saw in a co-translational process, but it also occurs to the mitochondria, to
the plastids and to the peroxisome. Usually, translocator protein complexes are
involved in the process of transmembrane transport.
- And then the third kind is vesicular transport, where vesicles move proteins
between the compartments. We’ll see examples of this when proteins move
from the ER to the Golgi and when the proteins move from the Golgi to the
endosomes or the lysosomes and also for proteins that are bound for the outside
of the cell in secretory vesicles.
Selective transport of macromolecules
Free diffusion of small molecules (<5000 Daltons)
- The first type is called gated transport and this refers to the movement of
proteins between the cytosol and the nucleus. This occurs through what is
called the nuclear pore complex. The nuclear pore complex traverses the two
membranes of the nucleus, so if you’ll recall the nucleus has two lipid bilayers
surrounding it and the nuclear pore complex goes through both of those
membranes and forms a pore.
- This pore is gated meaning that it is selective for the transport of
macromolecules, it is called a pore but it is selective for certain
macromolecules. However, if molecules are small enough, less than 5000 Daltons, they can freely move between the nucleus and the cytosol and the
nuclear pore complex is no longer selective against these molecules. Anything
above 5000 there is selective transport by the nuclear pore complex and
anything below 5000 can freely diffuse through the nuclear pore complex into
the nucleus or out of the nucleus.
From cytosol to nucleus
From nucleus to cytosol
- The nuclear pore complex can accommodate transport in both directions, that
means into the nucleus or out of the nucleus.
- Here is a diagram of a nuclear pore complex. There are approximately 30
proteins called nucleoporin that make up the nuclear pore complex, and this,
the whole complex you can see that it goes through both the outer nuclear
membrane and the inner nuclear membrane. Remember both of these would be
lipid bilayers, so there are two lipid bilayers that make up the nuclear envelope
that surrounds the nucleus.
- The nuclear pore complex can accommodate transport in both directions.
Nuclear import is the movement from the cytosol to the nucleus and nuclear
export is the movement from the nucleus to the cytosol.
- Some proteins need to move into the nucleus such as transcription factors
following their synthesis or their translation into the cytoplasm and then some
proteins need to actively be exported out of the nucleus so this can also be
some proteins that need to go into the nucleus sometimes but need to be
exported at other times during a certain event. Or you can also have ribosomal
proteins that are assembled in the nucleus and need to be translocated into the
cytosol in order to participate in translation. There are proteins that need to be
actively exported outside of the nucleus.
Binds to NLS (rich in Lys and Arg – these are positively charged AAs)
Binds to nucleoporins in NPC
- The import of proteins into the nucleus involves cargo proteins. The cargo
proteins are what are being delivered into the nucleus. It possesses a nuclear
localization signal or what is abbreviated as NLS. Nuclear localization signal is
a specific AA sequence on a cargo protein that will guide it or allow it to be
imported into the nucleus. Specifically, a nuclear import receptor will bind to
the nuclear localization sequence. The nuclear localization sequence is a
sequence that is rich in lysine and arginine amino acids. These are positively
charged amino acids.
- Once the NLS has bound to the nuclear import receptor, it binds to the
nucleoporins in the nuclear pore complex and then the cargo protein will be
transported into the nucleus.
- Here we have the cargo protein that is destined for the nucleus, it has the
nuclear localization signal rich in lysines and arginines, it will bind to a nuclear
import receptor and this nuclear import receptor carrying the cargo protein will
move into the nucleus.
- You can fuse different nuclear import signals to different proteins and they’ll
also go into different nuclear import receptors so you can swap. If you add this
protein or this NLS onto another protein that doesn’t normally have a nuclear
localization signal, you can guide it to go into the nucleus. This NLS is
sufficient to guide a protein into the nucleus. Binds to NES
Binds to nucleoporins in NPC
- Similarly, export of proteins out of the nucleus, the proteins that are going to
be shipped out of the nucleus are called cargo proteins. But in this case, you
have a nuclear export signal unlike the nuclear localization signal, it is the NES
nuclear export signal and he gave the examples of certain ribosomal subunits
need to be exported, RNA that is transcribed in the nucleus need to be exported
out, proteins with regulated nuclear import and export will also have nuclear
export signals on them.
- Similarly, the NES will bind to the nuclear export receptor. This is
structurally related to a nuclear import receptor but instead it binds to an NES
and then it binds to the nucleoporins in the nuclear pore complex and transports
the protein into the cytosol. Again you have a protein with an NES, this would
be the NES there, it can bind, this would be referred to as the cargo protein.
The cargo protein would bind to the nuclear export receptor through its NES
and this would be exported into the cytosol. Both the cargo protein and the
nuclear export receptor would be transported from the nucleus to the cytosol.
- Here is an overview of this process. On the left there you have nuclear import,
on the right, nuclear export.
- So cargo is being delivered in this case from the cytosol there to the nucleus
and you have the cargo there with the NLS, it binds to a nuclear import
receptor, both of them the cargo and the import receptor move into the nucleus
through the nuclear pore complex.
- Now what happens here is Ran-GTP is a GTPase that is required for this
nuclear import process. What will happen is Ran GTP will bind to the receptor,
that will release the cargo and the Ran GTP bound to the receptor will move
back to the cytosol that way the nuclear import receptor can be recycled back
to the cytosol. Once in the cytosol, the GTP that is bound to Ran will be
hydrolyzed to GDP so it becomes Ran GDP.
- We are going to go into more detail about how this actually happens but
basically just appreciate that Ran GTP plays an important role in the nuclear
- Similarly in the nuclear export process, here is the cargo protein with its
nuclear export sequence and in this case, the Ran GTPase bound to GTP will
bind together with the cargo protein to the nuclear export receptor. And then
this complex of Ran GTPase with the cargo and the nuclear export sequence,
nuclear export protein will all move to the cytosol through the nuclear pore
complex. What happens now is that Ran GTP, the GTP is hydrolyzed to Ran
GDP, it is released and so is the cargo and in this case, the nuclear export
receptor can just cycle back to the nucleus.
- From this slide it is important to recognize that in both export and import, the
Ran GTPase is required.
GDP bound form
GTP bound form
Stimulates GTP hydrolysis by Ran
Promotes exchange of GDP for GTP by Ran
- The Ran GTPase cycles between two forms, the GDP bound form and the
GTP bound form. You can see the GDP bound form in the slide and the GTP
form as well in the nucleus.
- The Ran GTPase is regulated by two proteins. One is called Ran GAP – GAP
stands for GTPase Activating Protein. What this does is it stimulates GTP hydrolysis by Ran. This results in Ran being bound to GDP. The Ran GTP is in
the slide, it moves out into the cytosol, Ran GAP activates the GTPase function
of Ran GTPase so Ran GTPase hydrolyzes GTP and you get Ran GDP. So the
Ran GAP is localized here in the cytosol and it activates or stimulates GTP
hydrolysis by Ran.
- On the other hand, in the nucleus you have Ran GEF which is a Guanine
nucleotide Exchange Factor and basically what this does is it promotes the
exchange of GDP for GTP by Ran. You’ll have Ran GDP coming into the
nucleus, it is acted upon by Ran GEF and then the GDP is exchanged for GTP
and you end up with Ran GTP bound in the nucleus.
- Now the differential distribution of these two regulatory proteins is critical for
maintaining the levels of Ran GTP and Ran GDP. Now Ran GAP is found in
the cytosol and Ran GEF is found in the nucleus. What happens is you get a
high Ran GTP concentration in the nucleus b/c Ran GEF is constantly
exchanging the GDP for GTP so the Ran GTPase is bound to a GTP in the
nucleus. However in the cytosol, Ran GAP is continuously stimulating the
GTPase activity of Ran GTPase, this results in GTP hydrolysis by Ran and Ran
GTPase becomes bound to GDP. So Ran GAP is converting Ran GTP to Ran
GDP and this is occurring in the cytosol. So that results in a high Ran GTP
concentration in the nucleus and a low Ran GTP concentration in the cytosol.
This is critical for the direction of transport as we’ll see.
- So Ran GTP we just mentioned that it is important for the direction of
transport and that is because Ran GTP moves to the cytosol with the nuclear
import and export receptors so this is shown in the slide.
- Let’s say this is a nuclear import receptor, once Ran GTP binds, there would
be an empty import receptor, when it binds to Ran GTP it is exported back to
the cytosol. There would be an export receptor in the slide there and when Ran
GTP binds with the cargo, it also exports out into the cytosol so in both cases
Ran GTP binding to the import or the export receptors result in the movement
of these receptors from the nucleus to the cytosol.
- Ran GTP once it gets into the cytosol, it gets acted on by Ran GAP which
leads to hydrolysis from GTP to GDP and you’ll end up with Ran GDP. Now
what happens is Ran GDP is cycled back to the nucleus and this involves
another protein called nuclear transport factor 2 or NTF2 shown in the slide
which is responsible for recruiting or transporting Ran GDP back to the
nucleus from the cytosol. Ran GDP moves back to the nucleus with the help of
NTF2 and then in the nucleus, Ran GEF will exchange the GDP for GTP and
then you get Ran GTP again and then this cycle can continue where Ran GTP
will bind to the nuclear import and the nuclear export receptors.
- Ran GDP and GTP are continuously moving back & forth b/w the nucleus &
Binds cargo in cytosol
Causes cargo release
Release of import receptor
- Now let’s look at nuclear import of cargo in greater detail.
- The first event occurs in the cytosol, there is an empty nuclear import
receptor, there is also a cargo protein with a nuclear localization sequence. First
event, the nuclear import receptor binds to the cargo, the receptor and the cargo
move to the nucleus through the nuclear pore complex.
- Now once in the nucleus this is where Ran GTP comes in, Ran GTP binds to
this complex and this causes release of the cargo shown in the slide. Ran GTP
takes the place of the cargo causing the release of the cargo and binds to the
nuclear import receptor. - Now once this is done, the cargo is now in the nucleus, the receptor has to be
recycled back to the cytoplasm so the empty import receptor with the Ran GTP
now moves to the cytosol.
- So then what happens in the cytosol is that Ran binding protein, this we didn’t
introduce but there is another protein involved in this association complex here
which is called Ran binding protein and Ran GAP which will lead to the
hydrolysis of GTP to GDP, both promote GTP hydrolysis of the Ran GTP and
release of the nuclear import receptor. Once this complex arise in the cytosol, it
is acted upon by two proteins, Ran binding protein and the Ran GAP and both
of these promote GTP hydrolysis so you end up with Ran GDP and that
releases the import receptor. Then what you get is an empty nuclear import
receptor that is free to bind to another cargo molecule and continue this
process. You can see this cycling.
- That was the nuclear import process so this animation will be available on the
Bio241 website you’ll want to take a look at it.
Binds Ran-GTP + cargo in nucleus
Release of cargo
Release of export receptor
- That was nuclear import so now we cover nuclear export which follows very
similar principles, in this case though you use a nuclear export receptor rather
than a nuclear import receptor.