Thursday January 29, 2009
- This is the next lecture, this is about sorting and packaging of proteins in
the Golgi apparatus.
- Just an overview, this is basically to remind us of how proteins destined for
the secretion pathway actually go through this process and we’ll see a little
movie of this in a bit. Basically all proteins or most of them are transcribed,
the genes are transcribed in the nucleus and then translated in the cytoplasm
and proteins that are destined for secretion are transported into the ER shown
on the slide.
- Thatnds the 1 stage, they get in there by transmembrane transport & then
the 2 stage is movement from the ER to the Golgi & this is through
vesicular transport so that is what we’re going to cover today, is how proteins
move from the ER to the Golgi through the Golgi & then sorted to different
compartments & we will focus on the lysosome today.
- Recognize or appreciate that both soluble proteins & transmembrane
proteins arstgoing through this secretion process.
- So the 1 destination from the ER is the Golgi and that is what we’re going
to cover today and this movie nicely shows what we learned so far about
secretion and where we’re going to.
Movie - Fluorescently labeled membrane proteins start their journey to the plasma
membrane after synthesis in the ER. They are first dispersed throughout the
extensive membrane network of the ER.
- From there they move to exit sites that form in random locations in the
membrane network. At each of these sites, the membrane proteins are
concentrated and packaged into transport vesicles. Clusters of the transport
vesicles fuse to form transport intermediates.
- At the next stage, transport intermediates move along microtubule tracts to
the Golgi apparatus near the center of the cell. The membrane proteins exit
the Golgi apparatus.
- They move in transport vesicles that are now pulled outward by the
microtubules which deliver them to the plasma membrane. Each time a Golgi
derived vesicle fuses with the PM, its content proteins disperse.
- That video nicely shows well first movement in the cell and how proteins
get through the secretion pathway so we’re going to start with the movement
from the ER to the Golgi.
- First just to give us an overview of what the Golgi looks like, we’re all
familiar with this structure but basically you would have the ER up there
and movement of proteins is going from the ER to the first part of the Golgi
which is called the cis Golgi.
- The Golgi can be separated into different cisternae, so these are different
layers of the Golgi. The first one that is encountered would be the cis Golgi
network or the CGN. That leads into the cis Golgi, this cis Golgi network is
the closest one to the ER and that would be called the cis face of the Golgi
that moves to the trans cisternae and eventually to this complex structure of
membranes and vesicles called the trans Golgi network. From there vesicles
or proteins are sorted to their appropriate compartments in the cell like the lysosome, the outside of the cell or the endosomes for example.
- That is the structure of the Golgi.
Bud from ER exit sites
Bound by cargo receptors
- This is part of the secretion pathway we covered today but how do these
vesicles bud off the ER?
- You will recall from the last lecture that the vesicles budding off the ER are
covered by a COP2 coat so these are COP2 coated vesicles budding off the
ER and they bud off at sites called exit sites on the ER. An exit site would be
a location where proteins are accumulating to enter into this vesicle and the
bud will form and the vesicle will form at this exit site. The cargo has to be
recruited to this exit site so it can be entered into the vesicle if it is destined
for the secretion pathway.
- There are signals called exit signals on many of the cargo that is going to be
in this vesicle that is budding off the ER and have these exit signals and
many of the soluble proteins have an exit signal that will be bound by cargo
receptors so there is an example there, that would be a cargo receptor, then a
soluble protein. You have an exit signal on the soluble protein that will bind
to the cargo receptor. The cargo receptor has an exit signal of its own and
that will allow it to be recruited to the COP2 coated newly formed vesicle or
a vesicle bud. Then these exit signals are thought to interact with specific
components of the COP2 coat.
- Those exit signals are found on soluble proteins, they can also be found on
transmembrane proteins, so for example, on a protein in the slide, it could be
a cargo receptor or it could be a transmembrane protein destined for a
different compartment in the secretion pathway, it would also have an exit
- Importantly, if proteins are not folded properly, they do not want to be
secreted through the secretion pathway so they are retained in the ER by
specific chaperones that will bind to unfolded proteins & prevent them from
being packaged into these newly formed COP2 coats that are going to the
- There are other cargo that don’t appear to have any of these exit signals,
we’re not going to go into the specifics of what these exit signals look like.
Some don’t have one and they are packaged into these newly formed vesicles
presumably based on their high concentration in the ER. Since they’re in
such high abundance in the ER, just naturally they’ll be accumulating in
these newly found vesicles. Then they’ll bud off with this vesicle.
Shed COPII coat
- Once a vesicle has formed, these are COP2 coated vesicles coming off the
ER, the coat will be shed and when the coat is shed, each of these vesicles
actually has a v-SNARE and a t-SNARE associated with it. On one
vesicle, you have both vesicular SNAREs and target SNAREs. Normally
these would be held together and coiled around each other, but the NSF
which is involved in mediating the uncoiling of these are associated with
the vesicle, also it keeps them separated and what this does is it allows the
newly formed vesicles from the ER to fuse with one another and form this
structure called the vesicular tubular cluster. So you can see, vesicles are coming from the ER, they shed their COP2 coat and through v-SNARE t-
SNARE interaction, these vesicles fuse together to form the vesicular
tubular cluster and this is actually found before the Golgi apparatus.
- The vesicular tubular cluster will move to the Golgi apparatus along the
microtubule network, these motor proteins are actually carrying the
vesicular tubular cluster to the cis Golgi network. The vesicular tubular
network is this network of tubules that is formed from the fusion of newly
formed vesicles that are budding off the ER. These will eventually move
to the Golgi apparatus.
Escaped ER resident proteins
Proteins involved in vesicle budding from ER
- In here, there are a lot of proteins that are destined to remain in the ER,
they need to get back to the ER so there is what is called a retrieval
transport. That’s the formation of vesicles from this vesicular tubular
cluster and these vesicles are COP1 coated vesicles, these ones that are
budding off the ER are called COP2, the ones that are in the retrieval
pathway are called the COP1 coated vesicles
- These vesicles are budding off the vesicular tubular cluster and there are
also some coming off the Golgi and they are moving back to the ER and
they’re bringing with them, escaped ER resident proteins, so proteins that
are destined to stay in the ER like the chaperones that bind to unfolded
proteins, and they’ll also include proteins that are involved in vesicle
budding from the ER so formation of these COP2 coated vesicles so
proteins that are specifically required for the formation of these coats will
also be brought back to the ER
- There needs to be this retrieval transport to make sure ER proteins or
proteins that only function in the ER don’t make it past into the Golgi and
into the secretion pathway so there is a way to bring them back. How is
Bound by KDEL receptor
Signal bound by COP1 coats
- ER resident proteins, many of them have what are called ER retrieval
signals on them. This is a protein sequence that will signal for that protein
to be retained in the ER and to go into these COPI coated vesicles of the
- Soluble proteins that are ER resident proteins have a signal called the
KDEL sequence, this is for the amino acid code that it encodes, so a
Lysine, aspartic acid, glutamic acid and a leucine. That is the amino acid
sequence that is found in these proteins. This sequence will be bound by
what is called a KDEL receptor so this is a receptor that actually binds to
this amino acid sequence and then this receptor, along with this soluble
ER resident protein will be packaged into this COP1 coat and then a
vesicle will bud off and go back to the ER and bring your ER resident
protein back to its appropriate place.
- Membrane proteins on the other hand have a very different sequence, it is
a KKXX sequence at the C terminus, these are two lysine residues
followed by two other variable AAs at the extreme C terminus of this
protein. This is an AA sequence found on ER resident membrane proteins.
One of these is actually the KDEL receptor so the KDEL receptor has this
sequence at its C terminus, so it will be packaged into the COP1 coated
vesicles. This KKXX sequence will bind to COP1 coats & that is how it is
recruited to these appropriate vesicles – these proteins will be packaged in
there & the membrane proteins will go back to the ER where they belong.
- So soluble proteins have a KDEL sequence, binds to the KDEL receptor. The membrane proteins like the KDEL receptor have a KKXX motif that
will package these into the COP1 coat & bring them back to the ER.
- So if you think about that, there is a problem because the protein has to
have, let’s say this is a chaperone protein, this soluble protein in the slide,
let’s say it is a soluble protein and is a chaperone protein, it needs to be
soluble in the endoplasmic reticulum, but then it needs to bind to the
KDEL receptor in the Golgi, but the KDEL is moving back and forth
between the vesicular tubular cluster and the ER. So how does this protein
get released once it gets back to the ER so that it can function normally?
High affinity for KDEL
Low affinity for KDEL
Binds in acidic pH; releases in neutral pH
- This is dependent on the pH in the different compartments, so basically
the pH gradually decreases as you move from the ER compartment,
through the vesicular tubular cluster to the Golgi apparatus – that means
that it is getting more acidic as you’re moving from the ER to the Golgi.
- The KDEL receptor cycles again b/w the ER & the Golgi but it needs to
bind to its cargo in the Golgi or the vesicular tubular cluster and then
release it once it gets to the ER.
- In the vesicular tubular clusters and the Golgi, there is a high affinity for
the KDEL sequence by this receptor, so that it will bind to the soluble
proteins that have the KDEL sequence.
- Once the receptor gets back to the ER, it now has low affinity for the
KDEL sequence in these soluble proteins and these proteins will be
released into the ER.
- Now this high affinity binding there (right), low affinity release there (left)
is mediated by the pH of these two compartments so basically, it will bind
at a more acidic pH to these KDEL sequences (soluble proteins) and it
will release them due to lower affinity for these KDEL sequences in the
more neutral or less acidic pHs such as those found in the ER.
- The pH of these compartments is regulated by a V-type ATPases which
will pump protons into these different compartments and decrease the pH
of these compartments so the more protons are pumped in, the lower the
pH will be, the more acidic the compartment will become.
- The KDEL receptor will bind in acidic pH such as those in the Golgi and
in the vesicular tubular cluster and release the KDEL proteins at the
neutral pH or more neutral pH that is found in the ER. So pH regulates
this release and binding/retrieval of these ER resident proteins.
Cycle between ER and Golgi, but transport to ER at slower rate
Prevent packaging into transport vesicles
- Now not all ER or Golgi resident proteins have these retrieval signals, but
they still end up in the right compartment, so not all ER proteins have
these signals so how do they stay in the ER or in some cases, some
proteins stay in the Golgi & never get taken back in the retrieval pathway.
- So there are different possible mechanisms for this.
- The first one is that there are different transport rates between the different
types of vesicles that are forming, remember that the ones going from the
ER to the Golgi are COP2 and going from the Golgi to the ER are COP