Bio 230 Lecture 9
Today we are going to look at how proteins get to their proper locations in the cell
mainly in eukaryotes since they have organelles. Recal that protein synthesis, the
translation by ribosomes occurs in the cytosol and once a protein is synthesized it
needs to get to the proper place, so for cytosolic proteins that is straight forward.
Remember that there is a bunch of organelles that are surrounded by membranes
in the cell and these membranes are not permeable to proteins so proteins can’t
diffuse in and out of these organelles so there needs to be specialized mechanisms
in order for proteins to get into where they are supposed to function. This is a
process called protein sorting.
Before we get into protein sorting lets look at review slides to make sure we are
on the same page.
Next week we will look at the import and export to the ER, and the ER that is
covered in ribosomes is called the rough ER and the ribosomes are synthesizing
proteins on the surface of the ER membrane. As they are synthesizing the
proteins, they are injecting the proteins cotranslationally into the ER (covered
Then we will see how the proteins move through the secretory pathway from the
ER to the Golgi and eventually end up outside the cell.
So here this is in an animal cell, this is in contrast to a plant cell that has a much
more uniformed structured look b/c it is surrounded by the cell wall. It is a rigid
wall that maintains the cells shape and allows the plant to grow quite large and
maintain the cells in a very strong conformation
Now another organelle that he wants to highlight in plants are the chloroplasts
which is the site of photosynthesis were plants convert light energy into chemical
energy and we will talk about how proteins get into the chloroplast
Another very prominent feature in plant cells is vacuoles; its filled with water but
plays an important role in terms of digesting macromolecules of the cell and
storage of many compounds
Highlight that the plant cell has all of the features that we talked about for animal
cells such as Golgi, ER, nucleurs etc.
When we talk about the cytoplasm it is the space outside the nucleus. In this case
the cytoplasm will include organelles so it is everything between the nucleus and
The cytosol is the aqueous part of the cytoplasm, it does not include the organelles
The lumen is the aqueous part inside the organelle. So organelles are surrounded
by a membrane and the liquid part is called the lumen.
Slide 6 Bio 230 Lecture 9
Protein sorting refers to the movement of proteins between various compartments
within the cell. It can be within the cell between different compartments (from the
ER to the Golgi for ex).
It can also be the movement of proteins out of the cell. When proteins go through
the secratory pathway, it enters in the ER and depending on what kind of signals
that that protein contains, that protein may end up outside the cell in a process
It can also be the movement of proteins into the cell and this is a process that we
will talk about in the second section of the course. This is the process of
It is important to remember that protein synthesis is initiated on ribosomes in the
cytosol. Remember that transcription is in the nucleus, mRNA is transported to
the cytosol and ribsosomes will translate in the cytosol and once they are
translated the proteins must be sorted to their proper locations.
There are 2 types of protein sorting;
Posttranslational protein sorting: This means that the protein is fully synthesized
in the cytosol before sorting to the proper organelle.
o This either occurs where the protein is unfolded, so the protein is actually
maintained unfolded in the cytosol following translation. There are certain
proteins that keep it unfolded and protect it from degradation. These
proteins can then be sorted to the mitochondria and plastids (these are
o Now this is in contrast to sorting using fully folded proteins and this
occurs for proteins that are going to the nucleus and to peroxisomes. So
the ribosome completely synthesizes the protein in the cytosol, that protein
folds and then it will be sorted.
The second category is cotranslational protein sorting: this occurs for proteins
that are destined to go to the ER. It is a specific type of protein, they need to have
an ER signal sequence at the end terminus. What happens is that the ribosome
initiates synthesis in the cytosol and the ribosomes direct it to the ER. Then the
ribosome is associated to the ER during protein synthesis. As the protein is being
synthesized it is being injected into the ER, this is cotranslational process b/c the
sorting is happening during translation.
We refer to 3 types of pathways by which proteins can be sorted.
o There is gated transport: refers to the movement of proteins between the
cytosol and the nucleus.
o There is transmembrane transport: this is movement of proteins from the
cytosol to the mitochondria, chloroplast, peroxixomes and ER. The key
here is that this type of transport requires protein translocators to move
proteins across the membranes that are surrounding the organelles.
o There is vesicular transport: in this movement of proteins its little vesicles
of lipid bilayers that move the proteins between different compartments. Bio 230 Lecture 9
What happens is a lipid bilayer vesicle buds off one membrane of a certain
compartment and will fuse with another compartment. As the bubble
forms it will carry a protein/set of proteins and when it fuses to the target
compartment it will release the proteins into that compartment. This
occurs between ER and Golgi and from Golgi into various compartments
including the cell exterior where proteins are moved by vesicles in the
process of vesicular transport.
Slide 9 and 10
Gated transport of proteins: protein movement between cytosol and nucleus:
The gate/major structure that is involved in the selectivity of proteins moving
between these compartments is called the nuclear pore complex.
The nuclear pore complex actually forms pores in the nuclear envelope and the
envelope is a series of lipid bilayers and again they will not allow passive
transport of proteins across. So the nuclear pore complexes form channels that
selectively allow specific proteins/macromolecules in and out of the nucleus
Its not all molecules that are selectively transported by the nuclear pore complex,
if the molecules is smaller 5000 daltons (most proteins are larger), these
molecules can move by diffusion in and out of the nucleus through the nuclear
The nuclear pore complex is actually selective only for macromolecules that are
larger then 5000 daltons
There are 2 processes that can occur: there can be movement of proteins in both
directions. The process of moving proteins from the cytosol to the nucleus is
called nuclear import through the nuclear pore complexes. The opposite of this is
nuclear export which is the movement of proteins from the nucleus to the cytosol.
Diagram bottom left slide 10: the nuclear envelope is 2 lipid bilayers that
surround the nucleus and the nuclear pore complex actually forms this channel
through the nuclear envelope. It is through the nuclear pore complex that proteins
will be translocated selectively, its not all proteins that are allowed to move
through this gate its only certain proteins that have certain signals on them. But it
is a channel, it is not completely closed so that is why small molecules are
allowed to move freely in and out of the nucleus.
Proteins that are moving into the nucleus during nuclear import are cargo
Cargo proteins have specific AA sequences on them called nuclear localization
Nuclear localization signals is a AA sequence that tells the cell to import that
protein into the nucleus
There is a nuclear import receptor that moves the protein from the cytosol into the
nucleus and it binds to this AA sequence on the cargo protein called the nuclear
Now the AA sequences are not a specific AA sequence but they are rick in lyseine
and argenines. These are positively charged AAs and those properties that allow
this sequence to bind to the nuclear import receptor. Bio 230 Lecture 9
Once the cargo is bound to the nuclear import receptor this complex will bind to
what are called nuclear porins in the nuclear pore complex. Nuclear porins are
protein molecules that are lining the nuclear pore complex. These are the proteins
that make the channel through the nuclear pore complex.
It is called the nuclear pore complex b/c it is not only one type of protein that
makes up this channel it’s a complex of proteins. One of the major players in
making this structure are nuclear porins
So once this has occurred the cargo has bound to the nuclear import receptors, the
nuclear import receptor is bound to these nuclear porins, then the receptor and the
cargo are translocated into the nucleus from the cytosol
This figure highlights that you can have different import receptors, there is not
only 1 type, there are different types that can regulate the movement of different
types of proteins and as mentioned this NLS sequence is not a specific AA
sequence, so they can vary and different NLS sequences can be recognized by
different nuclear importer receptors but they all carry out the same function.
In this case the protein are moving from the nucleus to the cytosol are once again
called cargo proteins
This time they have a nuclear export signal (NES), usually it’s a series of
hydrophobic AAs that vary in their sequences
What type of molcules have NES? Ribosomes, many of the subunits are
assembled in the nucleus, they need to function in the cytosol to carry translation,
so there is nuclear export of assembled ribosomal subunits from the nucleus into
the cytosol. mRNA molecules are transcribed in the nucleus and need to be
shipped into the cytosol to be translated by the ribosome so RNA are exported.
Also proteins where their movement between the nucleus and the cytosol is
regulated. Under some conditions they are in the cytosol and other conditions they
need to be imported into the nucleus. Ex coming..
So you have the cargo protein that needs to be moved and then you have a
receptor that is involved this time its called the nuclear export receptor. It is this
receptor that binds to the cargo protein, its structurally related to the nuclear
import receptor but they are different and they will bind in this case to the nuclear
export signal. So the receptor and the cargo move into the cytosol.
So recap: so you have cargo, binds to a specific receptor via a specific sequence
(nuclear localization sequence: NIS and NES) these complexes with the receptor
and the cargo move through the nuclear pore complex.
Slide 13 14
Now this whole process of import and export is maintained by a special class of
proteins called Ran GTPases and these are fundamental to the movement of
proteins in and out of the nucleus and they play a role in both aspects: import and
Ran GTPases are GTPases, it can hydrolyze GTP into GDP. So Ran GTPases
cycles between GDP bound form and the GTP bound form.
There are 2 classes of proteins that can regulate the Ran GTPase: one of them is
the Ran GAP: this is GTPase activating protein and this activates the GTPase
activity of Ran. So when that happens it stimulates GTP hydrolysis by Ran and Bio 230 Lecture 9
Ran will be bound to GDP. So Ran will be bound to GTP if the Ran GAP acts on
it, it will hydrolyze the GTP into GDP and will end up in the GDP bound form.
Now the second class of enzymes is called Ran GEF is a guanine nucleotide
exchange factor and what t