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Let's get started.
Hi, everybody.
My name's Chau and I'm with the Bill Lane Center.
The Center supports research on the American West and
advances our understanding of Western land and life.
Every year, we offer a number of summer
internships in the West.
There are opportunities in museum studies, environmental
conservation, publishing, state legislations, and more.
Each internship has a generous stipend and runs for 10 weeks.
You will have an opportunity to live and work in places
like Idaho, Montana, Colorado, and California.
The application deadline is February 7.
I left a printout of internship opportunities which
you can pick up after class, and you can find out more
information by visiting our website at west.stanford.edu.
Thank you.
[APPLAUSE]
OK, let's get started.
On Wednesday's lecture, we mainly talked about how to get
proteins into the ER and into the secretory pathway, how to
get the secretory pathway started.
And I think I've shown this cartoon several times so I'm
not going to tell you again, but just watch it.
Think about the biological processes.
What are the players here, and how are the nascent chain
targeted to the ER membrane?
What is the channel that the polypeptide chain is
transported through the ER membrane?
And we talked about the cycles.
Things are dissociated and then recycled for the next
round of protein synthesis.
We talked about the peptidase that cleaves the signal
sequence away, therefore resulting in a smaller mature
protein compared to what you would predict
from the DNA sequence.
And this is how secreted proteins or soluble proteins
are targeted to the ER lumen.
And just as a reminder, we have pretty much talked about
many of the sorting pathways, so how the free ribosomes
actually synthesize proteins that are targeted to the
nucleus, mitochondria, chloroplast, and peroxisome,
and then the ER associated ribosomes guide proteins into
the ER lumen.
And today, we're going to talk about these pathways.
What are the destinations?
Once the protein has reached the ER lumen,
what do they do next?
And just to make things a little bit more interesting
than you guys sitting here, I'm giving you a
quiz at this moment.
This doesn't count toward your scores, so no worries.
So let's say there's a protein, and from looking at
the sequence--
and I found that there are several of these targeting
sequences, including the signal sequence at the
N-terminus, and in the middle of the protein, there contains
a nuclear localization sequence, and at the very end
of the protein, it contains a peroxisome
targeting sequence one.
And we've talked about this.
This will guide the proteins on its own into peroxisome.
If there's a problem like this in the genome, where do you
think it will go?
Any takers?
You're smiling.
No, no, no.
You.
That's why you shouldn't smile.
Peroxisome.
Peroxisome.
OK, number one answer.
Any other answers?
Yes?
Gets secreted.
Gets secreted.
OK, number two.
Any other takers?
So I've listed the three sequences.
Now two are taken, so there's only one left.
So who thinks it's going to be in the nucleus?
Raise your hand.
No?
Wow.
There's a few hands, OK.
And who thinks it's going to be secreted, as he said?
OK.
And who thinks it's going to be in the peroxisome?
Right, OK.
So the right answer is it's going to be secreted.
Tell me why do you think it should be secreted?
The signal sequence must be translocated into the ER, and
then it goes to the Golgi apparatus but the nuclear
localization sequence and the peroxisome targeting sequences
don't get recognized.
OK.
So let me restate what he said.
So he said that the signal sequence is going to already
guide the peptides into the ER lumen, and therefore, the
other two signal targeting sequences are not going to
have even a chance to see the organelles.
So that is correct, but why is that the case?
So if the proteins are synthesized, it's sitting in
the cytosol, why wouldn't the nuclear localization sequence
be recognized by importin.
[INAUDIBLE].
That's right.
That's right, but that still didn't quite answer the
question why the importin will never have a chance to see the
nuclear localization sequence.
You raised your hands first.
It goes into the ER lumen before the sequence is even
translated.
Excellent.
[INAUDIBLE].
That's actually the core of the answer.
So the answer-- oops.
I didn't even actually put it up here.
So let me go back to the movie and remind you
exactly what happens.
Now we have a purpose when we look at this.
So now, when the signal sequence is first synthesized,
as we said, the translocation process in most of the
mammalian system is cotranslational.
So at this moment, the SRP binds to the signal sequence
and halts the translation.
So the translation is arrested.
So at this moment, the NLS and the PTS is not even made yet.
Before this translation could pursue or continue, the
peptide is already guided into the ER lumen.
So therefore, by the time that those nuclear localization
sequences are made, it automatically gets inserted
into the ER, so there's not even a chance for
importins to see them.
So that's the reason.
Yes?
So when do the other two sequences come into play?
Why do they exist at all?
They exist in proteins that do not have the signal sequence.
Yes, but why would they exist in that particular protein?
Oh.
Very good question.
Remember we talked about nuclear localization sequence.
What is the nature of nuclear localization sequence?
Do you remember what's actually in the sequence?
[INAUDIBLE].
It's K's and R's, right?
The K's and R's are everywhere in the genome, so they don't
necessarily mean that it is a sequence that
goes into the nucleus.
They exist.
The K's and R's are being used for many different ways, so
one way the cells read out the K's and R's are having the
importins bind them, and that sequence is only meaningful
when you don't have a signal sequence.
All right.
OK.
So now, let's start talking about--
is that clear?
Any other questions?
Yes?
Why would you have a nuclear localization sequence
[INAUDIBLE]?
Yeah.
As I said, these sequences, these are very short stretches
of amino acids.
They are lysines and arginines.
They don't have to be a nuclear localization sequence.
In other words, our prediction of nuclear localization
sequence doesn't necessarily mean that it acts to do that.
So basically, we know that if you cut off the signal
sequence and then force the cell to make this polypeptide
in the cytosol, then it won't be recognized by the importin.
So this is important.
I'm hearing some echoes.
So let's now move on to talk about what happens now.
The proteins are guided through the translocon into
the ER lumen and the proteins are folded.
So what's going to happen next?
So several things occur in the ER.
One of them is protein glycolsylation.
Glycosylation is the process where sugar moieties are added
onto the protein backbone.
This happens to many proteins in the ER.
Most of the proteins in the ER will actually go through this
process, and it often occurs on the asparagine residue site
chain, and usually the enzymes that actually transfer the
polysaccharide onto the asparagine, recognize a short
sequence aparagine--
and x means just about any amino acid except proline--
and then [INAUDIBLE]
on the next residue.
So the sugars are basically added onto this asparagine
side chain.
So most proteins get into the ER will actually get this
modification, and very few cell soluble proteins will get
glycosylated like this.
So it's a signature.
So you see a protein get glycosylated with this sugar
chain added, then it's a pretty good bet that it has
been in the ER.
In terms of the function of this gylcosylation, I have a
question mark here because it's not very well
established.
So you'd think that it should be pretty easy to do, and what
you need to do for that is to find a way to inhibit the
glycosylation reaction and then ask the question, what is
the consequence to the protein?
Does it hurt the protein?
Does the protein fail to target it to the right place,
or can they be functional?
And the answer is that when you inhibit, using drugs,
these glycosylation reactions, the protein seems still to be
able to target it to the right place and
maintain its function.
But what has been documented is that indeed, glycosylated
proteins show higher resistance to proteases.
So proteases, again, are enzymes that chop up proteins,
turn over proteins, degrade proteins.
So the idea is that when you're glycosylated, these
sugar chains are like this fence that are sticking out on
the surface of the protein, and therefore preventing or
making it harder for the protease to gain access to the
protein and chop it up.
But again, the functions are not super well established.
And some of the protein interactions depend on the
glycosylation.
We'll see examples of that later.
So we know that from George Palade's famous pulse chase
experiment, that after proteins or labeled proteins
are seen in the ER, the next stop that they make seems to
be in the Golgi apparatus.
So let's see exactly how this occurs.
It turned out that, as we said, the ER is an extended,
continuous memory compartment, meaning that the lumen of the
ER is actually continuous.
Newly synthesized proteins that got dumped into the ER
lumen can basically diffuse across the entire ER because
the lumen is continuous.
There's no barriers within the ER lumen.
But between ER and Golgi, the lumen, or the space, is
discontinuous, which means that the Golgi is a set of
membrane that are distinct.
It's a set of membrane compartments that are
discontinuous from the ER.
Therefore, to get proteins from the ER to the Golgi,
there has to be what's called vesicular transport that
mediates this movement.
So the vesicular transport has several different components.
Basically, it has to be formed from the ER structures.
So the ER buds out these vesicles, which contain the
components from the ER, both trans-membrane proteins as
well as lumenal proteins.
And then, these vesicles fuse with the Golgi structures, and
therefore becoming part of the Golgi.
So essentially, it pinches off from the ER, so it originated
from the ER.
So pinching off from the ER, naturally, the trans-membrane
proteins on that membrane came from the ER.
And it also has enclosed some of the lumenal
proteins from the ER.
And then these vesicles then migrate and move on, and then
fuse with the [INAUDIBLE]
component, becoming part of the Golgi structures, and
therefore deliver both the trans-membrane protein as well
as the lumenal protein, soluble, into the next
station, which is the Golgi structure.
So two key points here.
The traffic is two ways, meaning that there are
vesicles that go from the ER to the Golgi.
There are also vesicles that go from the
Golgi back to the ER.
So if the traffic is only one way, then at the end, of
course, the ER is going to continue to shrink and the
Golgi is going to continue to grow, and that's not what
happens in cells.
The cells have to maintain their ER
membranes and Golgi membranes.
And of course, the obvious dilemma here is that if the
traffic's two ways, how could you deliver specific proteins
from the ER to the Golgi?
That means the vesicles that move from the ER to the Golgi
are different from the vesicles that move from the
Golgi back to the ER.
If the retrograde signals now take up just the same thing,
then you don't get any net transport.
So these vesicles are different.
How are they different?
We're going to talk about in this lecture
and in the next lecture.
So when protein moves, their orientations is maintained.
So this is also very important, so this is the
slide to really illustrate that.
What it means is that here, the ER membrane is labeled in
two different colors, and the inner membrane, or the lumenal
side of the ER membrane, is labeled in light yellow, and
the cytosolic side of the ER membrane is labeled in blue.
And if you follow these two colors during this pinching
off and having the individual, isolated transport, or
intermediate vesicles and the fusion event with the Golgi,
you'll notice that the inner membrane or inner side of the
membrane becomes the inner side of Golgi.
So that means if you have a trans-membrane protein with
one side on the cytosolic side and the other side in the ER
lumen, when this protein, through this transport
process, got into the Golgi, the inside side of the ER
becomes the inner side of the Golgi compartment.
So that means the topology is maintained through this ER to
Golgi transport.
Why is this important?
This is important because in a similar way, the lumenal
protein in the ER becomes the lumenal protein in the Golgi.
And this is important because there are, let's say,
trans-membrane proteins that has an extracellular side that
sticks out of the cell and there's an intracellular side
that sticks into the cytosol.
These two parts of the proteins are doing very, very
different things.
So the extracellular part is actually interacting with
something outside of the cell, either binding to some
nutrient, anchoring the cell onto the extracellular matrix.
The solid part of the protein is doing
something entirely different.
It is probably doing some signal transduction rows, it's
talking to signaling molecules inside the cells, anchoring
the cells into the cytoskeletal
structures within the cell.
So they're doing very different things, so if you
get them mixed up or reversed, the molecule's
not going to work.
So this is a very stringent way of keeping the right side
of the protein to be exposed to the right locus.
And essentially, I mean, I don't want to too much into
how this is achieved, but the secret really lies between how
the vesicles, the two pieces of the membrane
fuse with each other.
This diagram shows that.
Basically, when the two membranes--
these are all lipid bilayers.
So they have their hydrophilic head pointing out and the
hydrophobic tail pointing inside.
And when the two membranes get very close to each other, the
fusion process dictates that the outer layer of the
membranes actually fuse first together and
then the inner membrane.
And therefore, the lumen from the one vesicle becomes
continuous space with the lumen of the recipient
compartment.
That's how the topology of proteins are maintained.
So now we know that the process that proteins take
from the ER to the Golgi is through pinching off some
membrane vesicles, and the vesicles move and seize the
Golgi compartment and then fuse with the Golgi
compartment.
But what are the proteins that do this?
All the proteins do this.
So all the proteins that are made from the
ER go to the Golgi.
You can think of this as a constituitive process.
So the ER is the protein factory that makes proteins,
and then they all got shipped to the next station, which is
the Golgi apparatus.
But of course, there are also proteins whose function is
inside the ER.
Can you think of any protein that actually needs
to be in the ER?
Translocons.
The translocons.
Excellent.
Any others?
Yes?
The signal peptidase.
The signal peptidase, yes, and then also the enzymes that
actually does that sugar modification.
So these are things that we've talked about that have unique
functions within the ER, so they need to stay in the ER.
And it turns out that they do that by getting retrieved or
scavenged through this KDEL receptor.
So these proteins that are ER residential proteins all have
a signature.
They have a four amino acid sequence at the very
C-terminus.
The most carboxy terminal of the proteins always end up
with this four amino acid stretch, KDEL.
These are four amino acid signature.
And then this sequence gets recognized by a receptor, and
this receptor is a trans-membrane protein that
localizes on the cis Golgi.
And then it collects these ER residential proteins and then
buds off a vesicle, and this vesicle, which compromise some
of the vesicles that go from the Golgi back to the ER, and
then dumps these protein cargoes back to the ER.
So it means that even for ER residential proteins where
their functions are in the ER, if you look at a steady state,
the predominant population of the proteins are in the ER,
but this polarized distribution is achieved
through a dynamic process in which the proteins that are
from the Golgi are constantly retrieved and dumped
back into the ER.
So it's a dynamic process, but it's efficient enough that
when you look at the steady state, most of the proteins
are in the ER.
Do the KDEL receptors tend to inactivate the bound enzymes
or proteins when that happens?
That's an interesting question.
I don't know exactly whether that has been tested, but what
I do know is that the KDEL receptor recycles.
So it basically needs to bind the cargoes here, but then it
needs to release the cargo, just like most of the other
trafficking scenarios.
So it does need to release the cargo back to the ER.
So I think, for proteins like signal peptidase, it probably
will have a problem if it's bound to the KDEL receptors.
Good question.
So now we've talked about the first destination of when
proteins get into the Golgi, some of these proteins contain
the KDEL sequence and they get shuffled
back to the ER structure.
What are the other protein destinations, and what exactly
is the Golgi structures, since we have already
talked about it?
And the Golgi structure is a stack of membrane disks.
Some people call it pancakes.
It looks like pancakes, I guess.
But it's a series of membrane disks that are stacked on top
of each other, and the vesicles, or the proteins go
from the ER to the cis Golgi, medial Golgi, trans Golgi, and
then to various destinations.
So these names are very confusing because if you read
different textbooks, they're even going to call them
differently, but in the next slide, I'm going to try to
find an easier way for you to remember what they all are.
It's actually not so important to really remember all these
names except really knowing that these are series of disks
and they're polarized structures.
So the disks that are close to the ER are actually different
from the disks that are far away from the ER.
This is a protein sorting factory.
Things happen while the proteins are migrating from
one disk to another disk.
And what's the function?
The function is that it's sorting and protein
maturation.
So the proteins get modified within Golgi.
There are sugars that are added there.
There are sugars are turned off from the Golgi.
We'll talk about that.
And also, proteins get sorted into different destinations.
So ER is sort of the post office of the cell.
So let's look at these disks.
So between the Golgi and the ER, in some textbooks, you'll
see that it's called the ERGIC structure, ER Golgi
Intermediate Disk.
And then migrate next is called the cis Golgi
structure, and that's a distinct set of disks.
And then the medial and the trans-Golgi are also called
the Golgi stacks, because they're really closely stacked
against each other.
And then this is actually a name that you need to
remember, which is the trans-Golgi network.
That means this is the
furthest from the ER structure.
And this is very important because this is where a lot of
the sorting occurs.
So this is the last stop before they become individual
vesicles that are destined to go to specific places, such as
plasma membrane secretion, endosomes, or lysosomes.
I think what I want to do is to show you guys--
I found this on the internet.
It's a nice cartoon that shows you this dynamic process.
So the vesicles are budded from the ER.
This is the stacks of Golgi structures.
So first, the vesicles that come from the ER will fuse to
form this, sometimes called cis cisterni, but then it's
basically the first set of disks.
And then here it's showing that within these structures,
there are enzymes that are modifying the proteins that
are going through it, making them become different.
And then the way that these proteins progress through the
Golgi structures is that this whole membrane stack, it
literally moves.
The original cis one becomes the medial one, the medial one
becomes the trans.
And then the new cis Golgi, or the ERGIC, is formed behind
the last one.
And then at this moment, in the trans-Golgi network, the
furthest away from the ER structures, the proteins are
getting sorted.
And they're getting sorted based on the signals that they
have within the protein sequence.
And then eventually, some of the proteins are being
collected over here, and then they bud off vesicles.
And these vesicles are different from these because
their lumenal or
trans-membrane cargoes are different.
These proteins contain different sorting signals,
therefore, they get sorted into different vesicular
populations.
And then these vesicles now do various different things.
Let's go back to my presentation here.
So in the cartoon, you already saw that there are proteins
modifications that occur through the Golgi.
One of the major modifications is, again, glycosylation.
And these are very complicated glycosylations that I can't
really memorize myself.
We said that the polysaccharide chains are
added onto the proteins, onto the arginine asparagine
residues in the ER, and these sugar chains are further
modified by a series of enzymes that
occurs in the Golgi.
For example, these mannose groups are chopped up first,
and then another moiety, the acidio-glucosamine is added
here, and then the other modifications.
There is further trimming, and then another residue is added.
And acetic acid is added at the end.
So it goes through a pretty complicated process of
trimming the sugars, adding new sugars to it.
And just two things that you should remember from this.
One is that these modifications, the specific
reactions, it's a series of modifications.
So the later modifications actually depend on the success
of the previous enzymatic reaction.
And then this occurs in a sequential fashion in distinct
compartments of Golgi.
So for example, this particular reaction that
involves trimming off these terminal mannose occurs in the
cis Golgi because the enzyme itself
resides within cis Golgi.
And then these three reactions occur in medial Golgi, and the
last reaction occurs in trans Golgi.
These reactions are so specific that if you see a
protein that got this modification but not this one
yet, you know that it has just made its way to the medial
Golgi but hasn't quite gotten to the trans-Golgi yet.
So these are signatures of being in a particular Golgi
compartment.
And the second is that there are specificities--
and we'll talk about this a lot in
the next set of slides--
and that means that some of the enzyme classes get
uniquely, modified, and they're sugar modified.
And it turns out that this sugar modification, at least
in this one case, has a lot to do with where these
proteins are going.
So sugar modification is one way of marking the proteins
and telling this group of proteins should go to one
destination.
We'll see exactly how that occurs.
So just a side note on these sugar modification enzymes.
So we've talked a lot about protein-protein interactions.
So for example, we've talked about the nuclear localization
sequence interacting with the importin, and we've talked
about KDEL sequence
interacting with KDEL receptor.
We've talked about the signal sequence interacting with the
SRP, the Signal Recognition Particle.
And in all these cases that we've talked about
protein-protein interactions, it seems that the proteins
always recognize a linear sequence of amino acids in the
target protein.
So for the NLS, it's a string of K's and R's.
For KDEL, it's KDEL.
For signal sequence, it's a bunch of hydrophobic amino
acids in terms of the protein.
So what this is that it tells you that it doesn't
have to be the case.
Proteins are folded structures, so they don't
exist in the single polypeptide chain.
They exist in the tertiary structures, quaternary
structures where they're folded.
In this case, the enzymes that recognize the protein targets
and then transfer sugar chains onto the protein target, this
is the enzyme.
The green stuff is the enzyme.
The enzyme actually doesn't recognize
a particular sequence.
It recognizes a patch, a domain of the protein, and
that came from a different part of the protein sequence.
If you follow the interface between the enzyme and the
substrate is this red surface on the substrate.
The red surface is not at a particular linear sequence,
and instead, it is one residue at the most N-terminus, and
then a little stretch over here and then a little stretch
over there.
Get the point?
So basically, these are two protein interaction surfaces,
and it interacts with here, here, here, and here.
It doesn't mean that in the protein sequence, they are
neighbors to each other.
So in this case, this surface is called the signal patch
instead of a targeting recognition sequence.
That's just a side note.
So we said sugar modification or glycosylation is one thing
that occurs within the ER.
The second thing that occurs in the ER is that protein got
proteolytically processed, and these are proteases that chop
up proteins.
And chopping up proteins doesn't always mean that it's
destroying the proteins because some of the proteins
are made as precursors, and then the mature product
requires proteolytically processing.
Meaning that, for example, there are hormones, and the
mating hormones, as well as a very important protein,
insulin, that regulates our blood sugar in humans, and
they're synthesized as polyproteins as precursors,
which means that if you look at their genes, the gene is
really large and the gene product contains multiple
copies of the mature protein.
Of course it has a signal peptide because it has to get
into the ER to be processed, but then within the Golgi--
not in the ER, but in the Golgi--
there are proteases that cut off these different copies.
And then there are N-terminal exopeptidase and then
C-terminal enzymes that trims off these little segments,
eventually giving rise to the mature proteins.
The mature proteins are often very short, only 13 amino
acids, very short compared to the initial precursors.
So there are these enzyme modifications that occur in
the Golgi that makes a person mature.
So that's the second thing that happens in the Golgi.
And the third thing that's very important for the
discussion of this lecture is the sorting
function of the Golgi.
So the Golgi serves as the post office of the cell.
And essentially, what it means is that-- from the cartoons,
you have probably gathered that everything
is made in the ER.
So regardless of whether the proteins are going to the
plasma membrane or the lysosome, they're
all made in the ER.
The ER is a factory.
It just makes proteins.
When it gets sent to the Golgi, in the trans Golgi
network, now the proteins are getting sorted into different
populations.
Let's say all the proteins that go to a particular
destination will end up in one patch of the trans-Golgi
network, and then when the membranes bud out from that
patch, then it makes a transport vesicle that are
going to different destinations.
And let's see one of the destinations from the Golgi,
and the first potential destination
is the Golgi itself.
Again, just like the ER residential proteins, there
are Golgi residential proteins.
They need to be in the Golgi because they are doing
specific things in the Golgi, and these things also, again,
have a very specific Golgi retention signal just like the
KDEL sequence.
We're not going to talk.
It's more heterogeneous than KDEL, but you get the point.
The concept is that having this Golgi retention signal is
going to be recognized by a retention receptor and these
retention receptors are helping to target these
vesicles to the right Golgi compartment.
I think my next slide is--
yes.
This was actually a little bit of a difficult concept for me
to initially get, so I want to spend some time with you.
So the current model is that we talked about how during the
protein maturation process, this whole
disk would move down.
The cis disk will become the medial disk, and then later
on, become the trans-Golgi disk.
So if the whole membrane disk is moving down the secretory
pathway, but at the meantime, we also said that there are
chemical reactions that only occur in the cis disk, only
occurs in the medial disk and the trans disk.
How could this be the case?
So when this whole disk is moving to the next station,
how could they maintain their compartmental specificity?
So it turns out that it occurs by,
again, vesicular transport.
So the cis disk, let's say, is defined by the enzymes that
are in the cis disk.
These are the sugar modification enzymes that are
characteristic of the cis.
And so then when the cis disk moves to become the next
medial disk, there's a sorting event that
already occurred here.
There are vesicles that are budded from the newly formed
medial disk that contains a lot of the enzymes that are
characteristic to the cis.
And then these vesicles fuse with the
newly formed cis disk.
So this is the way that, in a dynamic way, it maintains its
enzymatic specificity while shipping off the cargo to the
next station.
So if you didn't get this, I think the next movie will
really help.
The only way that I can really play only half of this is not
in the Display mode.
So now you can see that movement is occurring.
The cis is going to become the medial.
The old cis is going to become the new medial, and now the
new cis is going to form.
But then this still contains the enzymes that are defining
the cis, so therefore, the retrograde vesicle will ship
out those enzymes.
So in this way, it maintains the identity of this network.
Is that clear?
Yes?
In the video, there were never any [INAUDIBLE]
space proteins that [INAUDIBLE].
So is it only the membrane proteins that actually are
involved in the enzymes, or would there be some--
No.
These sugar modification enzymes are
actually soluble proteins.
So they also get budded off--
Yes, yes.
So again, this should raise a lot of intriguing questions.
So that means these enzymes must be collected to specific
patches of that membrane and this vesicle must bud off in a
very specific way and be targeted, and the vesicle
would know to fuse with the right compartment.
All these are very interesting, intriguing.
It can't be that simple.
The vesicle has to know where to go, which membranes to fuse
with in order to deliver the right cargo back to cis Golgi.
And all this is very interesting stuff, and we're
going to talk about some of that stuff next lecture.
What defines this fusion specificity?
What are the proteins that are actually getting the membranes
very close to fuse?
These are fascinating questions, and actually, this
year's Nobel prize is awarded to people who studied that
particular question.
So we're all going to delve into that a lot more in the
next lecture.
But for now, let's say we have one destination covered so far
from the Golgi.
One is to remain within the Golgi, and this is achieved
through sending some vesicles back into the Golgi.
And the proteins that are doing this contain a Golgi
retention signal.
And the second destination from the trans-Golgi network
is to go to the plasma membrane.
So this is, again, first illuminated by the George
Palade pulse chase experiment.
So the reactivity went from the ER to Golgi and then to
secretive vesicles on the membrane or being secreted
outside of the cell.
And it turned out that this is actually a default pathway, so
if there's no other sorting signals, the default pathways
send trans-membrane proteins or secreted proteins onto the
plasma membrane.
It gets a little tricky here.
So it's the default pathway if the protein contains the
signal sequence.
Agreed?
If it doesn't have a signal sequence, then
the default is where?
Cytosome.
Cytosome.
Very good.
Thank you.
So this is a default pathway.
The vesicles are generated from the trans-Golgi network,
and they're going to flow to the surface of the cell and
infuse with the plasma membrane.
Dumping its lumenal cargo, the trans-membrane protein becomes
part of the plasma membrane.
That's the default.
One thing has to be said here is that there are a second
population of the vesicles that are specialized, the
secretory vesicles.
These are actually, again, very important vesicles,
especially for particular cells.
So these default pathways, the secretory vesicles, are
constitutive.
So they occur all the time.
They're not very much regulated.
But in certain cells, like neurons, for example, the
synaptic vesicles are different in the sense that
their release is highly regulated.
So the release of the synaptic vesicles means that the
neurons are screaming at the next cells, get activated, or
get inhibited.
So this has to be really controlled by action
potentials.
You'll hear about that from Robert Sapolsky's lecture.
But it just means that there are other membrane organelles,
there are vesicles that come from the trans-Golgi network,
but they don't behave like the constitutive delivery pathway.
So these are fascinating questions.
They're incredibly important for neurobiology.
My lab is actually working on these.
There's very little that's known about what actually are
the sorting signals?
What are the mechanisms to sort these very important
synaptic vesicles?
But we just have to know there is a pathway like this, but
we're not going to discuss this further because
essentially, very little is known about it.
That's why we work on it.
And the fourth pathway that we're going to spend a lot of
time on is the lysosomal pathway.
And as I said, proteins that are going to the lysosomes has
the signature of receiving a particular sugar modification,
the mannose six phosphate.
And so this particular sugar modification serves as a
sorting signal to hoard these enzymes into the lysosomes.
So is just, again, giving you a view on
where things are going.
So out of the trans-Golgi network, there is a
constitutively secretion pathway, there's the
specialized secretory vesicles, and there are
vesicles that are targeted to the lysosomes, and then there
are also vesicles that are targeted to the endosomes, and
we'll see how that happens.
What are lysosomes?
Lysosmes are garbage factories.
Actually, that's probably not a right word for
it, recycling stations.
Proteins that need to be degraded get sent there.
In the life of cells, all proteins will have a lifespan,
and when they need to be degraded, they're sent to
degradation mechanism.
One of the degradation mechanisms is the lysosome
because it has a lot of hydrolytic enzymes that can
digest micromolecules.
And another very important source of material that gets
sent to the lysosomes are materials
that the cell acquires.
The cell is an entity.
It needs to eat things, it needs to drink things because
it has to ingest from its environment the nutrients, and
in order to grow, in order to differentiate, in order to
perform make new proteins, secrete new proteins.
So this eating process.
The eating process essentially involves endocytosing
micromolecules into the cell, and then this food for the
cell is sent to the lysosomes where there are enzymes to
chop them up, convert them into building blocks--
amino acids, lipids, sugar--
and then they can be used to build the cells.
So that's what happens in the lysosomes.
The lysosomes are small, membrane-bound vesicles, and
they contain a lot of hydrolytic enzymes.
They break down micromolecules and use as a component.
One of the signatures of the lysosome is
they're very acidic.
The pH is about five, not that acidic but reasonably acidic,
compared to the neutral pH in the cytosol.
It turns out that these enzymes are only active where
the pH is low.
And this low pH is achieved by a particular trans-membrane
protein, a proton pump on the lysosomal membrane.
This proton pump uses energies that are hydrolyzed from ATP
hydrolysis to pump protons into the lysosome.
And the more pumped protons you have, the lower the pH is
for this enclosed environment.
So now, we still have some time.
That's good.
So we can go into the details of how proteins are getting
sorted into the lysosome.
Here, we're going to talk about several things.
When you think about the sorting process, there are a
sequence of very interesting cell biological
phenomena that occurs.
One is that sorting implies that not every protein can go
into these vesicles.
Sorting implies that there has to be a selection process.
Some proteins collected here, and other proteins are ignored
or are not enriched in this patch of membrane.
So that's the first.
You have to collect the right targets.
And the right targets in this case means that the proteins
have to be specific because they receive this mannose six
phosphate modification.
This is the sorting signal.
If you get the mannose six phosphate, then you go into
this patch of the membrane.
That's the first, the target collection.
And second is that after the targets are collected, the
membrane has to deform to bud off, to pinch off a vesicle.
And then the vesicle eventually, the neck has to
pinch off so that the transport vesicles becomes
discontinuous from the donor membrane, the donor
compartment in this case, the trans Golgi network.
And then this vesicle has to, again, fuse
with the target membrane.
So let's look at some of these steps.
First is that, how do you collect cargo?
As I said, the mannose six phosphate is a sorting signal.
So enzymes or proteins, trans Golgi lumenal proteins that
receive this modification will get sorted into the lysosomes.
And it does so by binding to, again, a receptor.
So any biological signal has to be received or has to be
sent, has to be detected by a protein-protein interaction
through a receptor.
This is just another protein.
This receptor is a trans-membrane protein on the
trans Golgi network, and it has the two sides of things.
It has the Golgi lumenal side of the protein that combines
to the mannose six phosphate, the sorting signal, and it has
another cytosolic side of the protein that binds to
something called a clathrin and its adaptors.
The clathrin have heavy chains and light chains, and they
come together to form something called
a triskelion structure.
And this is the trimer, and the trimer will build up
eventually into a cage-like structure.
And it turns out that this cage structure is very
important to deform the membrane
and to form a vesicle.
So why do you need such a complex process
to deform the membrane?
It turned out that the membrane is the happiest when
the membrane surface is very low.
So it's like bubbles.
If you blow a bubble, they will form a specific shape.
To make small bubbles is actually very difficult
because the membrane wants to lie flat.
That's energetically most favorable, most stable.
So in order to form a bud off a membrane vesicle, it's
actually very difficult.
It consumes energy.
So these triskelion structures and polymerase structures
forms a cage.
This cage basically exerts the force that helps to bend the
membrane to form a vesicle structure.
But of course, there has to be links between this coat
structure--
or you can call it the coat-- the coat structure and the
target it's collecting.
So the goal here is to bend the membrane, but at the
meantime, collect a bunch of protein targets.
The secret here is in the adaptor proteins.
So the receptor protein, as I said, is a TGN, trans-Golgi
network, trans-membrane protein with its lumenal side
binding to the target that has received the mannose six
phosphate modification and with its cytosolic side
binding to the adaptor protein that links this
receptor to the cage.
Is that clear?
So just one more time.
Let's go from the most outside to the most inside of the
transport vesicles.
We're going to first see the cage-like structures formed by
the clathrin, and then the clathrin binds to the clathrin
adaptor proteins, and the adapter protein binds to the
cytosolic side of the mannose six phosphate receptor, and
then across the membranes and binds to the mannose six
phosphate modified targets.
So in this way, the clathrin coated--
this is called clathrin coated pits--
can collect the specific target proteins.
These adapters are sometimes also called adaptins.
It's just different names.
Here I'm showing you some of the EM structures just to show
you what it really looks like under Real Experimental Data.
You can see that over here, this is the lumen of the
protein, and you can see the outside of the protein.
And here, the key is to show you that the outside of the
protein has this coat, and they're decorated by an
incredibly regular array of proteins that you can almost
make out this little spokes that are coming out.
On a cross section, this very irregular
coat is made by clathrin.
Are these buds coming off continuously, and the clathrin
is coming off?
[INAUDIBLE] the protein, and it just comes
off every so often?
Or does the binding of the mannose six phosphate to the
mannose six phosphate receptor make that binding, the
clathrin to the [INAUDIBLE], more favorable?
That would be more efficient.
That's a great question.
Actually, the view in the field has switched from what
was originally thought as what you were referring to, a more
efficient pathway, to now, if you would believe, it's more
of a constituitive pathway.
So the clathrins are actually forming all the time, and then
it's collecting this.
I have a few more words to say on that, but
that's a great point.
Initially, people in that field thought that the binding
between the cargo and the adapter triggers the assembly
of the coat, but that view is definitely
changing at this moment.
So this is the EM pictures again.
So the inside is the cargo's being collected, and this is
really showing you the coat has very regular repeated
structures.
And this was even more striking on a scanning EM.
These are different EM technology where you'd spray
very fine metal powder on these purified vesicles, and
you can see that it generates great contrast on
the scanning EM.
It shows really the basket, the cage-like structures of
these coats.
In the next lecture, we'll talk about clathrin is
not the only coat.
These vesicles all different from each other, and one of
the defining differences is, what kind of
coat do they wear?
And the clathrin is one type of coat that
the cells can make.
Now we talked about the coat, and now what about the mannose
six phosphate receptors?
The receptors bind to these cargo proteins.
This allows this complex to be collected in this bud.
So now we talked about how the bud is generated, or the
budding event generates vesicles.
But the coat has to come off before the vesicles can fuse
with the recipient compartment.
And the coat actually needs to come off because there are
recognition processes that require the interaction
between membrane proteins over here and membrane
proteins over here.
The coat actually blocks that.
So the coat comes off, and there's some very
interesting-- again--
things that occur.
How do you regulate this coat?
And it turned out to be small G proteins again.
There are switches.
You turn it on, turn it off.
The code assembles and disassemble.
There are small G proteins involved in this process that
we're not going to really talk a whole lot about.
And then the vesicles, where do they fuse?
What's the recipient compartment?
The recipient compartment is a series of organelles called
endosomes, and the word "endo" comes from endocytosis.
When a cell eats stuff, it dumps the
stuff in these endosomes.
So these endosomes, on one hand, receives food that the
cell has just ingested.
On the other hand, it receives lysosome enzymes.
These are enzymes that degrade protein macromolecules.
So then when everything's getting here, the enzymes are
here, the substrates are here, it becomes a lysosome, and the
lysosome then degrades material.
If that's not absolutely clear, it'll be clear in the
next couple of slides.
The endosomes also contain low pH, and upon low pH, the
mannose six phosphate modified enzyme, its
receptor falls apart.
So the low pH sugars, the dissociation between these
sorting receptors and the cargoes and allowing the
sorting receptors to go back to the trans-Golgi network so
that it can be recycled and be ready for the next round.
And then the rest of the endosomes, as it receives more
and more of the hydrolytic enzymes that are delivered
through this pathway while receiving the macromolecules,
it matures.
This mature, it's not a very well-defined concept, but it
slowly becomes the lysosome.
So key point here is the mannose six phosphate
receptors move back and forth between the trans-Golgi
network and the endosomes, and then they're recycled.
They deliver the protein targets, which are the
hydrolytic enzymes, and then they're recycled back onto the
trans-Golgi networks.
And the cargo binds to the M6P modified targets in high pH.
So the trans-Golgi network has neutral pH, so
it favors the binding.
And the low pH in the lysosomes then triggers the
dissociation, therefore allowing the
release of the cargo.
The lysosomes are incredibly interesting and important
structures, and there are a series of human diseases that
are genetic diseases inherited that are caused by rare
mutations on enzymes that degrade particular things.
So the lysosome contains many enzymes.
There are enzymes that cut the sugar, there are enzymes that
cut the nucleotide, there are enzymes that
are proteolytic enzymes.
There are patients that have a single point of mutation in
one of those enzymes.
So actually, I've seen these patients.
One of our friends, actually, has--
very unfortunate--
two boys with a very rare disease
called Hunter's disease.
And these kids basically, every cell in their body is
missing one enzyme.
And the result is that the lysosomes cannot effectively
degrade one substrate.
And eventually, the lysosomes will expand, will become
really large because of the accumulation of the substrate
that cannot be degraded.
And their life expectancies are 10 years old, and it's
miserable because otherwise, they're developing all kinds
of problems.
They're usually very large and their bones
are actually deformed.
Their cognitive development is slowed down.
It's a devastating disease.
No treatment so far.
So very important stuff.
And then among the lysosomal diseases, the most severe form
is called the I-cell disease, and this is caused by a
mutation in this transferase that actually is critical to
put the mannose six phosphate onto the protein substrate.
You can imagine why this I-cell disease is actually the
most severe form of the lysosomal inclusion body
diseases because all the other diseases are missing one
enzyme that are cutting a particular substrate, but if
you have a problem with the mannose six phosphate
transferase, then essentially, all the hydrolytic enzymes
cannot undergo this sorting modification.
So then the lysosomes are missing all of
the lysosomal enzymes.
So this is actually is a very severe form.
The patients will usually die very quickly after birth.
So I just want to show you that this is very important
stuff that's related to medicine in many ways.
So now we've talked about this particular pathway.
The Golgi actually modifies its cargo proteins with this
modification and sends things into the lysosomes.
And then I've also already mentioned that the cell needs
to eat stuff, and then it needs to send also whatever
it's ingested from the environment to the lysosome to
be degraded or to be processed into building blocks that the
cells can use.
So exactly how do cells eat stuff?
There are small molecules, such as ions, sugar.
They go through channels and carriers
on the plasma membrane.
But large stuff cannot be doing this.
So macromolecules and larger structures are ingested
through pinocytosis and phagocytosis.
This is a picture of the active red blood cells eating
bacteria, and you can see that basically, there are
mechanisms that ingest the entire microorganism.
But another very important way that the cell eats is to use
this mechanism called the receptor mediated endocytosis.
And this process deals with macromolecules in the
extracellular space that are, again, ingested by the cells.
So essentially, the key point here is that the
macromolecules bind to the receptor on the cell surface
and then they get shuttled into the lysosomes and when
the lysosomes actually receive the enzymes from what we've
just talked about, then it becomes a lysosome and the
protein is, again, degraded.
So there are many examples and may cargoes go through this
receptor mediated endocytosis to get into the cell.
And we're going to just focus on two of the examples.
One is the uptake of the LDL, the low density lipoprotein
particles, and the second, we'll mention it later.
So again, this is very similar to the sorting between the
Golgi and lysosome in the sense that it uses adaptor
proteins and it uses clathrin coated vesicles.
So what is LDL?
LDL is the lipid particles that transport a lot of the
cholesterols in our blood.
So this is a topic that all of you have heard.
So basically, you go out tonight-- it's Friday--
treat yourself with a steak that's full of cholesterol.
The cholesterol is digested in your digestive tract and then
moves into the bloodstream.
And cholesterol is very hydrophobic.
It's lipid, and along with all the other lipids, very
hydrophobic.
How do the lipids move in your bloodstream, which is
otherwise aqueous solution, into the target cells, where
cells need the lipid to make organelles, to make membranes?
And this is the transport vehicle for cholesterol in
your bloodstream.
It basically is a protein lipid complex.
It has a lot of proteins on the surface, and it forms this
shell which then hides the cholesterol inside, creating a
pocket of a very hydrophobic environment so that the
cholesterol can be very happy inside.
And then this is essential.
It's very important for the cells.
This is the food how cells can actually
acquire or ingest lipid.
But of course, this is also bad for you and me because now
we're no longer in the jungle.
We're eating too much steak.
So the people with high levels of LDL in their blood tend to
develop problems.
This is cardiovascular problem.
Eventually, the wall of the blood vessels are damaged.
The blood vessels start building plugs on it, and
eventually, they get narrower and narrower so you have
stroke or heart attack.
So the concentration of blood LDL is a great indication, or
you need to control your blood LDL level if you don't want to
have heart problems in the future.
And it turned out that the LDL is collected, again, by LDL
receptors on the surface of the cell, and then the plasma
membrane buds off vesicles.
This is, again, utilizing clathrin coated pits, just
like the way that we talked about how Golgi sends enzymes
to the lysosome.
So there are adaptor proteins that bind to the receptor, and
the adaptins also bind to the clathrin, and this leads to
the generation of clathrin coated pits.
And the clathrin coated pits then shed off their coat, and
they deliver their components to the early endosome, and
then these early endosomes receive components from the
enzymes from the Golgi and then becomes late endosomes,
and then mature into lysosomes,
and lysosome degrades.
Just as a side note, this is worked out by two MDs that had
profound impact on our understandings of endocytosis.
And they were MDs.
This is Brown and Goldstein Joseph Goldstein and Mike
Brown at UT Southwestern.
They were both physicians that were interested in
understanding why there are genetic families of patients
and they get heart attacks in their 30s.
So essentially, the general population gets heart attacks
in their 50s, 60s, and 70s.
It takes a long time to for the high levels of LDLs in the
blood to clog up the blood vessels in people.
It takes decades for the disease to develop.
But then there are rare patients, it comes in
families, and they develop these cardiovascular problems
in their 20s, and these patients have heart attacks in
their early 30s and they die.
So they were interested in what's wrong with these
patients, so they took fibroblast cells from these
patients, and they did this endocytosis assay.
They added LDLs to these cells, and they found that
compared to the normal individuals, the LDLs are not
uptaken efficiently at all from these patients.
And then they figured out essentially what's wrong.
The mutations of these patients are in the receptors
and in the endocytosis pathways.
So this really gave them the entryway into trying to
understand the receptor mediated endocytosis pathway,
and they also established the correlation between the high
blood lipid and these cardiovascular diseases, which
eventually led to all these drugs--
Lipitor.
You've heard of probably some of them that controls the
blood lipid level, and that has really saved a lot of
people's lives, in my opinion.
Let me go through this slide more carefully, and then we'll
stop here and pick up from the next lecture.
More things to be said here is again, similar to the Golgi to
lysosome pathways, the vesicle has to lose its clathrin coat
before it can fuse with the target membrane.
And then the empty receptors most likely are returned to
the plasma membrane for the next round of endocytosis.
And then the early endosomes receive enzymes and becomes
the late endosomes, and then they're degraded.
In the next couple of slides, I think it's going to get a
little too complicated, but let me just end with this
video here that summarizes, if you wish, the life of a
secreted protein.
So here the red, this is the messenger RNA, and you can see
that the messenger RNA is getting
found by the ribosomes.
Just think about the processes.
The messenger RNA, ribosome is recycled, so then it makes
these proteins.
And this is the ER lumen, and the ER lumen is
the continuous space.
So the proteins are diffused across the ER space and get
collected and then forms these ER to Golgi vesicles.
And through vesicular fusion and budding, they go through
the secretory pathways onto the next stage, which is the
Golgi, and there are a bunch of membrane transport within
the Golgi apparatus between the stacks that maintain the
identity of the cis, medial, and trans stack while shipping
off the protein cargoes to the next station.
At the trans-Golgi network, proteins get sorted.
The constitutive secretory pathways send them off to the
plasma membrane, dumping its secreted into
the exocellular space.
And then there are endocytosis pathways that deliver the
ingested macromolecules to endosomes, and the endosomes
receive the hydrolytic enzymes and, with the low pH, become
degradation machines that give rise to the
building blocks of cells.
Thank you.
[APPLAUSE]