BARBARA IMPERIALI: I always
like to just remind you that the sixth -- it's kind of an assignment,
but the numbers-- we're going to do this
news brief project, where it's a teamwork
project if you choose. If you take a look at the piece
that you have in your hands now, it asks you for a
little bit of information on that, who you're
going to be working with, if you choose to
work with someone. Or you can work on your own. That's fine. And we're looking to
get a news brief that's of significance to research
going on in the life sciences. And I've given you-- there
are a couple of links in the sidebar of the website,
so good places where you can find interesting material. What I'm super
interested in for you, as a group where many of you
are in the engineering fields, is to find something really
cool at the interface between the life
sciences and engineering, where engineering has a huge
impact on the life sciences. You have alternatives. You can download the
coordinates of a protein and print it on a 3D
printer and give us a summary of what the
protein is, what it does, and submit your 3D print. I'll give it back
to you afterwards, once we've had a look at it. But actually submit
the 3D print. And then the other
opportunity is-- I think you'll
remember back to when we were talking about
molecular biology of the cell. I did kind of a clunky
demo at the front of the class, nothing like
Professor Martin's demos at all. This was me with the
ethernet cables showing you what topoisomerase did. But in my demo,
I didn't show you how topo also cuts
a strand of DNA, holds it while the
supercoiling unwinds, and then stitches it together. So I thought some
of the engineers might be able to
come up was something that was really better
than that for me to use, for us to use,
next year in class. So I'm really laying
down the challenge there. So I always like
things in the news. I thought this was
kind of interesting that the first vertebrates
evolved in shallow waters. I thought those were really
cool first vertebrates. I'd love to get one of them
in a fish tank and keep it. But anyway, that's that. It's truly amazing what
you can see in the science reports, news briefs. I look at them
whenever they come in. I get the posts every
two or three days. And I'm kind of pleased
to see that there's a lot of things that
are in those news briefs that I feel that we're
enabling you to read with some appreciation
because of what we're covering in the class. So what we're doing now is we're
really taking a leap forward here into cells and
organisms, with respect to understanding how
structure and function of individual macromolecules,
proteins, nucleic acids, sugars, determine life,
determine the dynamics of life that are necessary for
an organism to really go through a life cycle,
divide, have cells divide, go forward, have cells move. So what we're
going to be talking about in the next lectures,
which is section 6, is cellular trafficking
and signaling. And so for the first lecture,
which is 19 that we're on now-- so we're past the midway mark-- I'm going to be talking
about trafficking. And that is how,
within a cell, things get to where they need to be, or
they get exported from a cell. Because all of the
actions of a cell-- I really like thinking
about the cell as a circuit board, where
there's a receiver that gets information. And then the complex circuitry
determines what outcome you get at the end of the day. So many of the proteins
that we've talked about need to be in specific places
for the cell to function. We have to have DNA
polymerase in the nucleus. It's not going to be
useful in the cytoplasm. We have to have a transcription
factor that helps transcription go to the nucleus at the
right time for transcription to occur. But we don't want it
there all the time, because otherwise you'd
have the light switch on the entire time. That wouldn't be useful. So we need to regulate where
certain macromolecules are. We need to have the receivers
on the surface of the cell to receive signals from outside. This is not just pertinent
for multicellular organisms. It's pertinent for
unicellular organisms, for them to sense
their environment, know what's going
on around them. Is the salt
concentration changing? Is it getting very hot? Is it getting cold? Is there enough oxygen? Even unicellular organisms need
to receive signals and respond to them. Multicellular organisms
are way more complicated. Because you need
to establish organs and different parts of
a multicellular organism that have specialized function. So trafficking
really is about what happens after you've made a
replicated DNA in the nucleus, transcribed it, made a mature
messenger that goes out to the cytoplasm in most cases. We'll talk about the
exceptions to that case. And then in the cytoplasm,
when proteins are expressed, all the different
things that happen that guarantee that the protein
gets to a proper destination for function. And some of those are
quite complicated. Because remember,
if I'm going to park a receiver in the
cellular membrane with the signals being
captured from outside, I've got to get
from the cytoplasm out there in a reliable way. In lectures 20 and
21, I'll talk to you about cellular signaling with
a focus on mammalian cells and the sorts of
signaling processes that may go awry in cells, for
example, proliferating cells. And then Professor
Martin will really focus in on neuronal cells,
optogenetics in lecture 22. So this bundle really allows
you to call in the things that you've learned
until now and apply them into much more intriguing
and complex situations. So here's a wonderful,
sort of silly drawing of a triangular cell. There's always a joke
in cell biologists, when they're trying to
talk to mathematicians and mathematicians want
to simplify everything. And so everything
gets-- imagine a cell, and there's this box
shows up on a screen. Well, we all know
that cells aren't triangular or box-shaped. But nevertheless, I thought
this one was particularly cool. And so trafficking, the
process of trafficking, is really all about, where
is the information encoded into the protein that ensures
that the protein is where it needs to be for the
dynamics that we observe in living system? We've talked a lot
about static things. We make the protein. Here's the protein. The protein folds. We've talked a lot about
things that are kind of fixed in time and space. But what we want
to do is understand what makes a cell programmed
to undergo a new function. For example, something as
simple as cell division, we have to orchestrate a huge
variety of activities in order for the cell division
process to start to occur. Something as really simple a
cell mobility, think about, how do cells move? They're not moving all the
time, but sometimes they will move towards a signal. What triggers that
kind of interactions? So in looking at the cell, these
are some of the older images, where certain
organelles, for example, are stained so that
you can see them. So peroxisomes are where
degradation happens. The golgi and the ER
are a part of what's known as the
endomembrane system. You'll see a lot about
this towards the later part of the class, where
we talk about how things get outside the cell
through the endomembrane system. There's the surface
plasma membrane. The cytoplasm is this sort
of not really aqueous-- it's an open space. But it really isn't open. It's highly congested
with all kinds of molecules, all kinds of
structural proteins and so on. So don't think of the
cytoplasm as a solution, but think of it as a much more
gel-like structure with a lot of things happening in it. The nucleus itself is also
surrounded by a membrane, as is the endomembrane system. So this would be the
nuclear envelope. Within the nucleus,
you have a structure called the nucleolus,
where aspects of the nucleic acids necessary
for protein biosynthesis are made. Then there are
structural proteins like microtubules and actin. But now, in this day
and age, we don't have to deal with
these vanilla images. We can actually use
the methods that you've learned about in the last
section, recombinant biology, to create new versions
of proteins that have along with their sequence
a marker that gives them a fluorescence-colored marker. So we are, later
on in the semester, going to spend three lectures
on fluorescence and cellular imaging, where you'll learn more
about these fabulous proteins beyond just saying we've got
a green one and a red one. We're going to give you all
the background on the protein engineering that enabled those
to become tools for biology. But for now, I'm just
going to show you how much more interesting
the images of the subcellular structures are when
you've labeled, for example, a particular
protein that goes exclusively to the nucleolus with a
blue fluorescent protein, or to the mitochondria. Remember, Professor
Martin told you we always think of
these as-- and I'm not going to do the push-up. I'm just going to say it,
powerhouse of the cell. I'm not doing-- [LAUGHS]
I'm not great with push-ups, to be honest. But you see these sort of more
tangled, extended structures. Vimentin is more of
a structural protein. Here are the golgi, the
endoplasmic reticulum, and the nucleus. So the colored
fluorophore proteins, or the fluorescent
fluorophore proteins, actually allow us, in real
time, to observe dynamics. Once a protein is
made, where does it go? If we add a trigger to the
cell to cause an interaction, can we observe that
protein, for example, migrating to the
plasma membrane. Can we watch proteins
being made through the ER? A variety of different things
that allow us in modern biology to really look at dynamics,
not just static information. And so what I'm going
to talk to you about is the ways in
which proteins are coded very early on in their
genesis, in their biogenesis, in order to go to certain
locales within the cell. So let me just give you a bit of
a road map here with a protein. And where things may start-- so we have some options. Do we want to send the
protein outside the cell or keep it inside the cell? Obviously, two big default
differences, if you're going to go to a particular
venue inside the cell. Are we going to just
stay in the cytosol? That's a sort of simple-- actually, that is
the default position. Because you want to
remember that most proteins are made on ribosomes in
the cytosol of the cell. But the statistics are
that about 50% of proteins end up somewhere else
than the cytoplasm. They may end up in
an organelle, back in the nucleus on the
surface, or secreted. So there's a lot-- so it's a good, solid
50% that don't end up staying in the cytosol, where
they were originally made. Their alternative is
to go to organelles. And if you're going to
an organelle, remember, the ribosome is not membrane. It doesn't have a
membrane perimeter. But many of the organelles
do have membrane perimeters. So we're talking here
about the mitochondria. That is far too long of a word. The nucleus-- so I'm
going to abbreviate things like peroxisomes, or various
membrane-bordered organelles, where we're going to have
to figure out, if something is made in the
cytoplasm, how does it get into those organelles? Now we've spoken a
little bit about the fact that some proteins are
made in the mitochondria. I'm going to get back
to that in a moment. But all the proteins
in the mitochondria are not made in
the mitochondria. Some of them are shipped in. Remember the thing the
endosymbiont theory, where we said that
mitochondria may have originated from bacteria
and been engulfed into cells. Those bacteria obviously were
originally self-sufficient. But a lot of the proteins
that were expressed in the mitochondria
were dispensed with, and mitochondria
now use proteins that are encoded by
the nuclear DNA rather than the mitochondrial. But to this day, some
proteins remain encoded within the mitochondria. So these are opportunities
for where that may be. And I'm going to talk very
specifically about signals that can get proteins
into the mitochondria and into the nucleus. And it turns out
that the barriers around those organelles
are pretty different. I'll come back to
that in a second when we get on the next slide. With respect to going
outside the cell, there are two options. One option is for the protein
to remain in the plasma membrane but with part of its
structure outside the cell. So the other option is
for the protein actually to be spit out of the cell
as a soluble entity that can travel around an organism,
for example, in the bloodstream and go to a remote site. And that becomes very
important in signaling. So we would call those
proteins secreted and soluble. So these would be
membrane-bound. These would end up
being soluble proteins. Let's take a look at the
structure of the cell and look at where these
various components are. So if you see these dots,
those are free ribosomes in the cytoplasm. They would start to
express different proteins. A lot of proteins are
expressed in the ribosome. But in some cases,
proteins become expressed on ribosomes
that are associated with the endoplasmic reticulum. And therefore, you
start a process whereby proteins
end up being shipped to the outside of the cell. So where you see
the speckles here, the free ribosome,
and then the ribosomes bound to the rough
endoplasmic reticulum, here, your destinies are on
the right-hand side of that picture. And here, the destiny
of these proteins ends up on the left-hand side
of this sort of family tree that I'm showing you. There's obviously one more
place where proteins are made, and that's in the mitochondria. And if you remember the
first question on your exam, it described the DNA
that's in the mitochondria. Going back to the
endosymbiont theory, that's a circular piece of DNA. And it sets it apart. And the ribosomes
in the mitochondria look more like bacterial
ribosomes than you eukaryotic ribosomes. So remember, all
along, we're going to try in the second
half of the course to bring back knowledge
we've taught you, but sort of, in a
sense, endlessly remind you to keep the
big picture in mind. Because we've already
spoken to you about it. So this now is a
nice pictorial vision of what I've just
described to you. And I'm going to first of
all talk about proteins that are made in the
cytoplasm and may be shipped to
various organelles, and how that's accomplished. And then in the second
part of the class, I'll talk about how proteins
are shipped to cell surface, or through expulsion
from the cell. So the key mechanisms
whereby proteins are trafficked to new
locations are first of all using targeting
sequences that are part of the protein sequence. And this is a very common way in
which proteins are trafficked. They are part of the sequence. They may be at the amino
or the carboxy terminus. But they are woven into the
structure of your protein. So your protein comes along
with a barcode saying where it's going to necessarily end up. And for the nucleus
mitochondria and peroxisomes, for example, people
have done extensive work with bioinformatics to basically
look up protein sequences and find common themes
of particular sequences that may be common to where
a set of proteins may end up. Sometimes those
sequences may not be easy to see just
at first glance. But now there are websites
that you can very, very readily put your protein sequence into
the web site, and it will say, it's got a nuclear
localization sequence, or a mitochondrial-targeting
sequence. So we can either do
this by eye or we can use informatics analysis. Informatics analysis
is very valuable because sometimes information
may be a bit more encrypted. And it may be a real
struggle to slog through a lot of sequences. So you can really find out
about the targeting sequences through bioinformatics. Because nowadays, the genomes
of dozens and thousands of organisms are
available readily online. And you can literally
parse out information from the genomic
information that gives you the proteomic information. So that's one way, so with
sequences that are targeted. In some cases, those
targeting sequences remain part of the protein. But in other cases,
in order to ensure that the protein stays put,
the targeting sequences are removed. So that's another
important point. You may keep the
targeting sequence, or you may lose it through the
action of another enzyme that cuts off the targeting
sequence when destination has been reached. Now, there's a second
way that we can program where a protein may go. And these are rather
useful transformations that make things even more dynamic. So let me walk you
through a concept. If you think of a protein
that's made on the ribosome, it's got a targeting sequence. In order to get that
protein to destination, you've got to make a new
batch of protein that's going to go to its destination. It's going to end up
in the mitochondria. You've got to make
the protein de novo. Sometimes when we need to
have the action of a cell we can't wait that long. We can do things quickly
and expect the cell to suddenly change
what it's doing. Because we're sitting around
waiting for the ribosome to make new copies
of the protein. So the second way
in which proteins are targeted to
new destinations is through what's known
as post-translational modifications. This is so unfair, Adam. I saw you using
the middle boards, but it looked so much easier. So the second way to target
a protein to a destination is using post-translational
modification. What does this mean? What it means is that
the protein is made. It's ready. It's waiting. But we haven't engaged
its final destiny. We haven't triggered it to
go where it needs to be. But we're waiting for an
enzyme to just carry out a seemingly minor
modification of that protein. And then the protein
will go to its destiny. And I've shown you
here examples of three types of modifications. One we will talk about today,
because it's very simple to understand, lipidation. And then the other
two, we'll talk about next time, phosphorylation
and ubiquitination. And these are all what
are known as PTMs, Post-Translational
Modifications. And they are changes that
occur to an amino acid side chain within an already made
protein to alter its destiny. And I'd like to talk
about lipidation first, because I get to remind
you about cellular membranes. So remember, we've talked about
these semipermeable barriers that are around organelles
and around cells. And let's say that
this is a membrane-- I've got to put my-- that exists between
the cytoplasm and the outside of a cell. And let's say I have a
protein lurking around in the cytoplasm, but I
need it at the membrane. I need it to get involved
in a signaling process. And I need it now to be there. If I have a soluble
protein, it's not associated with the membrane. But I can use another
enzyme to attach a hydrophobic, greasy
tail to that protein. So what it really
wants to do is to get to the hydrophobic membrane. Lipidation is such
a modification. It's just the modification with
a long-chain, often C16, C18, fatty acid that then renders
the protein lipophilic and makes it want to move, and
insert this lipophilic tail into the membrane, and part the
protein of the plasma membrane. So the information
is still, though, encoded within the protein. How could that happen? How could I have made that
information be in the protein? What might be the
strategy there? It's still encoded,
but it's secret. It's cryptic. Any ideas? So I'm not going to just glom
this group onto a protein. I'm going to put it
somewhere specific. And so oftentimes,
lipidation reactions occur site-specifically
at particular sites within a sequence, and an
enzyme recognizes that site and transfers the
lipidic molecule to it. So lipidation actually
may occur, for example, of the amino terminus
of a protein. But if there are certain
features within that protein, you may then attach
the lipidic group. So once again, using
bioinformatics, you can look at the
target protein of interest and predict that it's the
target of a post-translational modification reaction. So once again,
the information is programmed into the sequence,
but it's quite cryptic. It could be within the
middle of the sequence. There could only maybe
be a couple of clues. But the clues are
there nonetheless that can be parsed out using
computer learning and screening of sequences to say that
is a target for lipidation, or phosphorylation or such. Is that clear to people? Does that make sense? The information is encoded, but
you can't see that it's there. But the advantage of
the post-translational modifications is that
they occur on demand, as opposed to making
a new protein de novo, and then having it go to a
particular cellular location. Later on, when we talk
about phosphorylation, you will see that
phosphorylation is the bread and butter
of cellular signaling. It's the light
switch in every room in the cell that turns on and
off in order to make functions happen within the cell. And that's a really major,
dynamic post-translational modification that has
significant meaning. So the reason on
this little image-- I just wanted to
show you the membrane and just remind you that the
membrane is a supramolecular structure that's assembled
with a hydrophobic core and polar head
groups on both faces, as I've sort of indicated
in this cartoon. So let's start
with sequences that might take us to the nucleus. Now, the nuclear membrane
is rather a strange entity. Because the nuclear membrane
isn't a simple membrane like the plasma membrane. It's actually a
double-layered membrane. So if you look at a
nuclear membrane-- and I'm just going to do a job
of showing a portion of the nuclear membrane. Within the nuclear
membrane, there are pores, quite
launch openings. And the membrane is actually
a double membrane, where all of these lipid bilayers. So it's not a single membrane. It's a double membrane
with large openings. And you might say,
well, that's no use. There's just these great big,
gaping holes in the nucleus. Anything can come
and go if it wants. But the nuclear pores are
kind of a special structure. Because they have a protein
that's kind of disordered, that creates a tangled network. That means that that
pore isn't totally open, but there's some
stuff that something's got to get through to get
from one side to the other. And my colleague Thomas
Schwartz in biology works on the macromolecular
structure of nuclear pores to understand
these structures. Because these are also
made through the auspices of having a lot of proteins
that help create this structure. Otherwise, that
membrane wouldn't stay in its proper format. So in order for a protein
to get into the nucleus, if it needs to, or
leave the nucleus, it has to have some
kind of mechanism to get through this
structure that's plugging the nuclear pore. So this would be the
inside of the nucleus. And this would be the cytoplasm. So as shown on this
slide, the nucleus, there's a particular
protein sequence that's appended to a protein. That's known as the Nuclear
Localization Sequence, or NLS. And what an NLS
sequence is, it's a short sequence
of amino acids that enables a protein to get
to its proper destination. And these sequences are
quite well recognized. They may end up being
highly basic sequences. So an example of an
NLS would be Lys-- it's not very
exciting, but it just goes on, Lys, Lys,
Lys, arginine, lysine. And it may be bounded
by hydrophobic residues or other types. So that would be a typical NLS
sequence that's in a protein. And I want to remind you that
lysine and arginine all have side chains that
at physiological pH are positively charged. So the nuclear
localization sequence is something that's
easily recognized because of this sort of
short sequence that may be at the N- or C-terminus. I think there's
either possibility. But it's a very clear sequence. You could look at your
protein sequence and say, there's an NLS on that sequence. And it's the NLS
sequence alone that's responsible for
getting the proteins in and out of the nuclear pore. Let's mostly focus on
getting into the nucleus. Basically, you have
a protein structure that has an NLS sequence
at one terminus. And that NLS sequence
binds to another protein. Creatively, you had a
little bit of chance to give proteins
names in the exam. It's called importin. So it's an import protein
that binds to the NLS, and as a consequence of
that, will carry cargo. It will escort cargo into
the nucleus of the cell. And it sends it through
this meshwork of proteins. That's a very loose
mesh work of proteins. And they're not
ordered proteins. They're highly
disordered proteins. So they make more of
a filter than a plug. But they are
definitely something that doesn't allow
any old protein to go through that nuclear pore. NLS tags are very easy
to recognize, once again, through bioinformatics analysis. And what's really
cool is that you can reprogram a
protein to be where you want by manipulating the NLS. So this is rather a
nice set of experiments. Let's say we have a protein
that we're going to micro-inject into the cytoplasm of the cell. And we want to program it to
either go into the nucleus or stay outside the nucleus. That can be done
readily by attaching a nuclear localization
sequence to a protein along with a fluorophore dye
or fluorescent protein that will allow you to
observe that experiment. If you micro-inject
into the cytoplasm, that protein that's got an NLS
will get run into the nucleus through association of
the NLS with importin. But if you chop that NLS,
the protein the stuck, remains out in the cytoplasm. Let's say you want to
study a new protein. I just want to show
you that these NLS sequence are totally independent
of the cargo they carry. You can just stick an NLS
on your favorite protein who you want to interrogate. Let's take pyruvate kinase. It doesn't have anything to
do with specific transport to the nucleus. But nevertheless, if you put-- if it doesn't have an NLS,
it's fluorescently labeled, it stays outside
in the cytoplasm. But if you put an
analysis on it, you concentrate into
that region of the cell. So these experiments
show you that what we know about these
targeting sequences can be manipulated and used
to enable you to move things around in the cell. So that's one particular
type of mechanism. The next mechanism I
want to describe to you is the mechanism that's used
for mitochondrial transports. And it's a little bit
different in its strategy. So to get into the mitochondria,
there is, again, a recognition sequence, in this case, a
mitochondrial localization sequence that has
particular characteristics. In this case, the mitochondrial
localization sequence, let's say it's at the
N-terminus of your protein. And it would be something that
might be a mix of charges. Some Arg, Glu, Arg, Glu. So that's a typical
MLS sequence. And in this case, the
charge at physiological pH is different from the nuclear
localization sequence, because it's an alternating
positive and negative charge. So this is pretty
different from this. It doesn't say bioinformatics
to figure that one out. So you can then pick out
mitochondrial localization sequences. And so in this case,
remember, mitochondria make some of their own
proteins on their circular DNA. But they've abandoned
expressing all the proteins that are needed in the mitochondria. And some proteins
are transported into the mitochondria using
these types of sequences. But the approach,
the strategy, is different from getting
into the nucleus. In this case, the MLS
sequence associates with a protein channel
that is in a closed state. So here's a membrane. Here's the makings of a channel. But it's in a closed state. But once the protein with the
NLS sequence binds to that, that channel opens. It's triggered by the
binding of that sequence to a portion of the protein
that's outside that membrane. And that then allows the protein
to be unfolded and transported into the mitochondria, where
that sequence may be removed. And then protein refolds
in the mitochondria. So it's a very
different strategy for that and the nuclear
localization sequence. So you'll find, for many
different organelles in the cell, there might be very
specific localization sequences that you could look
up and learn about. But one thing I want
to mention to you is that these localization
details are very important. And many diseases in
cells are a consequence of proteins not being
localized to the right place. If you're not in the right
place at the right time, then things will
start to go wrong with the signaling or the
processes of the cell. So diseases are
frequently associated with mislocalization. So now what we're going
to do is basically say, we've taken care of
understanding things made in the cell. They either stay in
the cytosol or they'll go to organelles based on
particular types of strategies that are largely dependent
on short tagging sequences, but in other cases, may be
dependent on post translational modification. All right. So here is a cartoon. But actually, I want to do
something slightly different if it doesn't take too long. Now, when we first
talked about translation on the ribosome, what you
see there in green and yellow is the ribosome. The dark band is
a messenger RNA. The dark blue are
transfer RNAs that are being helped with elongation
factors to get to the ribosome. But what I want
to point out here is the emerging
sequence of polypeptide coming out through a
tunnel on the ribosome. Now, if a protein is going to
be destined outside the cell, it is expressed with what's
known as a signal sequence. It's about a
20-amino acid residue sequence that is recognized
by the signal recognition particle. And then translation
slows down and clamps the ribosome on the
endoplasmic reticulum membrane so that the
new peptide starts being threaded into the
endoplasmic reticulum through what's known
as the translocon. So you're now not sending the
protein out to the cytoplasm, but you're rather
sending the protein into the endoplasmic reticulum. And you're also sending it
down this branch of the protein biosynthesis pathway. You see this piece
of protein emerging. This hatched portion
is the cytoplasm. The gray portion is the
endoplasmic reticulum. So there is a complex
machinery at play that enables proteins to
be made in the cytoplasm but now targeted to a
completely new location. And these are the
proteins that are going to be destined to either
stay in the plasma membrane or be secreted from the cell. And this view here gives
you a little bit more than the cartoon. So ribosome-- a
signal peptide is made that is a green
peptide sequence that's about 20 amino acids long. That is actually called
a signal peptide. It's signaling for
synthesis through the endomembrane network. That causes the ribosomes to
dock down on the cytosol ER membrane and keep
on being synthesized so that proteins are made
into that endomembrane system. And you can think of this
cavernous endomembrane system as your tunnels out
of a cell for either display on the surface of the cell
or for secretion entirely in vesicles. So let's take a look
at how that occurs. When you make a
protein in that way, see the dark dots, the rough ER? These are ribosomes that are
attached to the membrane. Proteins are made
into the membrane. And then the endomembrane
system is not really just a tunnel or a labyrinth. But actually, each
of those layers spits off vesicles that
fuse with next layers to gradually make their
way outside of the cells. So here you see
there are vesicles. You're always keeping
proteins associated with membrane as you go through
the endomembrane system. And here is a vesicle
that's got protein in it. It may either release it
to the outside of the cell, or the protein may be
associated with the membrane of the vesicle and stay
parked in the plasma membrane. And so I just want to give
you one final slide where I talk about the biogenesis
of membrane proteins. Now, this is pretty
complicated stuff. Because you have to remember
what's inside and out. So I spent more time than I
should have on this cartoon to show you which
end of the protein ends up outside the cell
and which inside the cell, and how you make
multi-membrane-spanning proteins. So let's take a look at this
in detail now, looking-- here's the ribosome. Here's the protein emerging. If there's signal
sequence there, that ribosome docks
down on the membrane and starts translating the
protein, amino terminus first, into the
endoplasmic reticulum. We'll all OK with that. As synthesis continues, we
may reach the stop codon on the messenger RNA. And what may happen is
that the protein may remain associated with membrane. The amino terminus
will be in the ER. And the C-terminus will
remain on the other side. There are a number of
different configurations. But if we want to start
to transport this protein to the surface of the
cell, that will then stay associated with
membrane but not in the form of the flat membrane
that it was delivered into. But that membrane may pinch
off into a spherical vesicle. But you still have
the C-terminus outside and the N-terminus inside. That will then work
its way through the endomembrane system,
and ultimately, fuse with the cytosol. This is the really fun part. And then, once it's
fused with the cytosol, it has the option
to be displayed on the outside of the cell. Why? You have a protein. The N-terminus is
on the outside. The C-terminus is on the inside. So that shows you the
biogenesis of the cell surface protein that's stuck
in the membrane through its
membrane-associated domain. If you're not going to
stay with the membrane, you can actually also
simply release this into the vesicle for release
of a soluble protein. I will not go through this. But there are
miraculous steps that end up in the biogenesis of
multi-transmembrane proteins. Because each of those
transmembrane domains gets made in the translocon
and gets shuttled sideways. And you start piling up
transmembrane domains that span the membrane. And in the next
class, we're going to see how useful these proteins
are in cellular signaling. So those are very important
proteins to think about. One last thing-- so
let's think about this. For either configuration, either
post-translational modification or using targeting sequences,
when do we define where the protein's going to end up? Where's the information
first defined? Anyone want to answer
me and explain why? Yes? AUDIENCE: Would it be
B, the mRNA sequence, because that would have
a significant portion of the splicing? BARBARA IMPERIALI:
It's a good try. But you want to remember,
yes, splicing is important. But when was the
sequence actually in the entire pre-mRNA? When would that
have been defined? Yeah? Sorry. Carmen? AUDIENCE: Is it in the
genomic DNA sequence? BARBARA IMPERIALI: Yes. Because you never have
information in the RNA that wasn't in the DNA. So the DNA has got
the information there. Yeah, it may need a bit
of splicing to put things in the right place. But the information
is there in the DNA. So you want to remember, for all
of this targeting information, it's in the genomic
information most commonly. It's the genomic
information that has the patterns of sequences
for post-translational modification. It's the genomic
information that has things like NLSes and MLSes. They're already there. But they are often encrypted. And there was a very
nice point there, though. If you want to send to make a
single chunk of a genome that encodes either a protein
that's going to be exported through the secretory pathway
or stay in the cytosol, you might splice in or
out a signal sequence. So that's a really good way,
using the same original DNA sequence, to actually
get to proteins that fulfill different final
destinies within the cell. So next time, we're going
to talk about signaling. It's going to be a blast.
