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Good morning. Good morning.
Today I'm going to talk about,

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this is lecture 14, and I'm going
to talk about protein localization.

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Now, some of you may remember
that earlier in the semester I was

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walking around with this sling.
And so to help me from writing on

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the board, even though it is
this arm, I have made a PowerPoint

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presentation of most of the
things that I would have written

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on the board. And for
your ease and comfort,

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this PowerPoint presentation will
be posted online so you won't have to

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write down everything in my slides.
So just sort of sit back. And I

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will write a few things on the
board, so you can write those down.

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OK. So now you guys have heard
about central dogma from Professor

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Eric Lander and you've heard
about gene regulation last Friday.

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Here is something that
you're familiar with,

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this image here
depicting central dogma.

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DNA replicates to DNA. This
is replication. Replication.

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DNA is translated, excuse
me, transcribed to RNA.

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Transcription. And RNA
is translated to protein.

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Central dogma. Where does this
occur? Where does replication

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occur in a cell?
Nucleus. Good.

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Where does transcription
occur in a cell? Nucleus.

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I heard nucleus. That is
correct. And where does

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translation occur?
Cytoplasm. Ah, and yet we know

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that these processes
require proteins to do them.

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OK. You've just described
where these processes occur in a

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eukaryotic cell. Let's
say you're a bacterium.

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In bacteria, where does replication,
transcription and translation occur?

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Where? Cytoplasm. OK. So now
we've made bacteria look very

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simple. But they're not that simple.
And so let's take a look here.

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Here's a bacterial cell.
I've drawn what could be E.

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coli. I has an outer membrane, an
inner membrane, and the space in

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between is the perisplasm.
Now, here is its circular

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chromosome. I've transcribed some
gene to an RNA and a ribosomal pop

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on and make a protein
which his in the cytoplasm.

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Yet some proteins are
localized to the inner membrane,

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others are localized to the
periplasm, and some are localized to

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the outer membrane, and
others are actually exported

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completely outside the cell.
Even more complicated is a

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eukaryotic protein, because
not only does it have a

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plasma membrane where proteins
are localized. It has a bunch

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of organelles. There's
the nucleus and there's

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mitochondria and there's endoplasmic
reticulum and Golgi apparatus.

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And it, too, translates RNA
by ribosomes in the cytoplasm.

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So how do these proteins get
back to the nucleus or go into the

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mitochondria or get into the
organelles? So what we're going to

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do in the next few slides is
we're going to follow the process,

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because they're so similar in
bacteria and eukaryotic cells,

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of how proteins get to the membrane
and how they get outside the cell.

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And then I'll go back and talk about
how proteins get into some of the

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organelles. So let me show you
what some of the proteins are.

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So an example of a cytoplasmic
protein in bacteria is beta

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galactosidase.
You've heard about it.

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It breaks down lactose. It's
in the cytoplasm. And example

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of a membrane protein
is a lactose receptor.

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The lacY permease that's on the
surface of the cell brings lactose

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in. An example of a fully
secreted protein is a toxin.

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For instance, bacillus
anthracis makes anthrax toxin.

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It's completely exported from the
cell. In a eukaryotic cell there's

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a bunch of cytoplasmic proteins.
There are all of the glycolytic

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enzymes. And, for
instance, biosynthetic amino

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acid enzymes like histidine
synthesis enzymes, those

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are cytoplasmic. For a
membrane protein there are

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receptors, like the receptor
for insulin, a hormone,

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a peptide hormone, or growth factor
receptors, every receptor that's

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membrane-bound. And a
fully secreted protein.

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Some cells like pancreatic
cells secrete insulin.

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Some cells like some of your
immune cells secrete antibodies.

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OK. So it was not clear how these
cytoplasmically made proteins,

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proteins that were made in the
cytoplasm got to this location.

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And the person who worked
on this was George Palade.

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And this was in the fifties.
And he studied pancreatic cells

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because they're master secretors.
And he was able to perfect his

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microscopic technique. And
you can see here this is a

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pancreatic cell. This
is endoplasmic reticulum

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studded with ribosomes.
These are mitochondria.

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This is the nucleus. Here is
another picture that he took.

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And here is the rough endoplasmic
reticulum studded with ribosomes.

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Here's Golgi apparatus. And then
there are like little vesicles.

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So he did this experiment where
he decided he would pulse label

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proteins as they were being
synthesized in a pancreas,

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directly in a pancreas. So
what he did was he injected

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radioactive amino acids directly
into the pancreas of hamsters.

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I guess I could
draw a little

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hamster here. And he directly
injected radioactive isotopes.

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And what he's doing is these
radioactive amino acids will be

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incorporated into proteins
as they're being translated,

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and he can follow the population of
freshly translated proteins through

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the cell. So he injects hamsters
with the radioactive amino acids.

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And then at various time points he
adds, he also injects glutaraldehyde.

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So first the label, then
glutaraldehyde. And what this

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does is it fixes the cells in its
tracks. Whatever the cell is doing

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it just stops. And he
removes the pancreas and he

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looks at the cells.
This is fixes the cells.

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So, Tom, I don't know what's
going on here. Can we not use this,

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Tom? All right. It's just doing it
on its own. It has some time thing?

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Oh. All right. So what he found
was at the early time points,

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now, what I did, I did this, OK?
He didn't see yellow. What I did

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was I added yellow to his original
slide to show you at the earliest

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time point he found the label
associated with the endoplasmic

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reticulum. At the next
time point he found the

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label associated with the Golgi
apparatus. And then at even later

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time points he found the
label in secretory vesicles.

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So this is my representation
of what he found.

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So here's a cell,
nucleus, mitochondria.

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The early time points the label
was in the ER followed by the Golgi.

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I didn't do that.
Yeah, take it out.

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Followed by vesicles.
It's not working. OK.

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Sorry for that. I'm sorry. So
he won a Nobel Prize for this

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work because it is the pathway
that still holds true today.

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Now, he won the Nobel Prize in '74.
And at about that time, maybe '71,

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another scientist names Cesar
Milstein was working on immunology.

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And what he did was he fused
a cancer cell with a cell that

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constantly secreted antibodies,
so he ended up having an

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immortalized antibody producing cell.
And he was doing some research and

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he did in vitro analysis. And
he found that antibodies that

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were produced in vitro were longer
than the ones that were actually

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coming out of the cell. So
he proposed that there was an

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N-terminal end. He
looked and saw that the

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N-terminus was different,
and he proposed that it would

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possibly be cleaved upon export.
OK? And so this is, he won a Nobel

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Prize, not for this work but
for his work in immunology.

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And this was also correct. And
so this is from his lecture,

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Nobel lecture. It says in vitro
synthesis of immunoglobulin light

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chains, that's what he was doing,
to our delight we ran into the

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unexpected observation of the
existence of a biosynthetic

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precursor of light chain.
Further experiments led us to

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propose the extra N-terminal
sequence was a signal for vectorial

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transport across the membrane
during protein synthesis.

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This was the first evidence that
indicated the signal for secretion

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was an N-terminal segment rapidly
cleaved upon protein synthesis.

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OK. So now there was a student
of Palade. He was a post-doctoral

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student. His name
was Guther Blobel.

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And he saw this experiment in 1971.
And he thought how do we know it's

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not an artifact of in vitro science?
Well, how do you know that the

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ribosomes are not just hoping
on earlier in the message?

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Maybe that's why it's a longer
protein. And he didn't buy it.

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So he wanted to further pursue
this. And he did it in the

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Palade manner. So what
he did was he took a test

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tube and he added message
for exported protein,

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ribosomes and charged tRNAs. And
when I say charged tRNAs I mean

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tRNAs that have amino acids attached
to them. And so if you add those

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three things to a test tube you
find a protein is made. So then

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he added microsomes.
What are microsomes?

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OK. We're going
to pause this.

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Now, I told you what Palade found
out. He found out that proteins

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were first seen in the rough ER in
the lumen. This is the lumen right

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here. This is where he
first observed radioactivity.

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Now, if you take endoplasmic
reticulum and you share it,

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it forms little tiny vesicles,
little vesicles with ribosomes on

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the outside. And they're
called microsomes for

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small things. Microsomes.
And they're essentially little

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rough endoplasmic reticulum vesicles.
So when Blobel added to the same

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test tube that had the RNA,
the ribosomes, the tRNA.

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When he added microsomes he found
that the protein was still in the

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supernatant. There was no protein
found in the lumen of the microsomes.

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So he figured, OK,
it needs something.

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Let me go extract something
from the cytoplasm.

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And he extracted lots of fractions.
And he added these cytoplasmic

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fractions. And he found that one
faction actually was able to cause

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the peptide to enter the microsome.
And if he added this fraction late

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in the reaction, the protein
would never get into the

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lumen of the microsome. But
if he added the fraction late,

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I mean, excuse me, early, if he
added the faction early they would

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get in. So let me just summarize.
So message ribosomes, tRNA, you

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find protein in the supernatant.
Message ribosomes, tRNAs, plus

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microsomes, protein in the
supernatant, not in the microsomes.

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You add a fraction that works
sometimes, but if it's added late

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the protein is in the supernatant.
But if you add that fraction early

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the protein is in the lumen of the
microsomes. So he interpreted this

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result as that there was an amino
acid sequence at the beginning of an

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exported protein. And that's
recognized by a complex

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that was in the fraction. This
complex is required to get the

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protein to the lumen of the ER.
And to get to the lumen of the ER

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the protein has to be just
beginning to be translated.

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Now, since not all proteins
have the same N-terminus,

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Blobel predicated, like Milstein,
whatever the sequence was it would

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later be cleaved. And he
won a Nobel Prize for this

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in 1999. And it wasn't just for
this, because he went on and he

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actually figured out
the entire pathway.

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In the next few slides I'm going
to show you what he discerned.

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One thing I want to just point out,
though, the experiment he did was

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heterologous. So the
extract came from wheat germ,

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the microsomes came from dog, and
yet it still worked. And it was

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right because this pathway is
universal. And let me show you how

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universal. It's used in bacteria
and it's used in eukaryotic cells.

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So here's a bacterium,
it's translating a message.

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Here's the signal starting to be
translated, it's an exported protein.

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The same thing, in the
cytoplasm a signal sequence

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is being newly made. And
here's a close look of the

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signal sequence. It's
about 20 amino acids long.

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It has a couple of positive
charges at its extreme N-terminus.

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In the middle there's about seven
to twelve hydrophobic amino acids

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variable. And this called
a signal sequence. OK,

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so let's take a look at what happens.
OK. Now we're in the cytoplasm of

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a eukaryotic cell. Here is
a signal sequence emerging

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from a ribosome here.
What recognizes it is SRP.

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That's what he named his complex
for signal recognition particle.

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So SRP binds to the signal sequence.
And, if you recall, it takes it to

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the ER to be translated.
Here's a picture of the ER.

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And there's a docking protein or
SRP receptor. So the SRP binds to

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the docking protein, it
brings with it the signal

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sequence which is attached to the
ribosome, which is attached to the

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message. Adjacent to the docking
protein is a translocon which is a

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channel composed of proteins.
The ribosome pops onto the

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translocon. The SRP floats away.
And notice that the signal sequence

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is in the membrane, excuse
me, starts to enter through

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the membrane and
translation resumes.

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The signal sequence is cleaved by
a signal peptidase within the ER,

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it cleaves off the signal
and translation continues.

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And if it's a fully secreted
protein it's fully internalized

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within the lumen of the ER
and the ribosome pops up.

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If it's a membrane protein
the signal is cleaved again,

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translation resumes, and then
it gets imbedded in the membrane.

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And so I'm going to shut this off,
otherwise it's going to keep going

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on its own here. So if
it's a membrane protein what

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does it have? It has a
transmembrane stretch.

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You've seen this maybe before
in problem sets. So it's a

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transmembrane stretch.

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Or transmembrane domain. It's
about 20, 22 amino acids long.

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It can be 30 maybe. So we'll
say 20 to 25 amino acids of

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hydrophobic residues.

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It is a stop transfer sequence.
Stop transfer for going across an

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ER membrane. It anchors
it in the membrane.

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It forms
alpha helix.

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If this part is
the lumen of the ER

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right in here then
this is the cytoplasm.

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OK. So that's
how membranes and

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proteins look of this kind.
Now, as you can see here,

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it's in the membrane. It's
imbedded in the membrane.

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And I've drawn a different membrane
protein over here because this

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protein is going to work its way
to the far side of the endoplasmic

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reticulum. OK? As the
fully secreted protein will

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also work its way. In the
endoplasmic reticulum sugars

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get put on these proteins. So
when they get to the far side

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they bleb off into
little transport vesicles.

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OK? So here's the cytoplasmic
protein completely within the lumen

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of the vesicle. Here's
the membrane protein

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imbedded in the membrane of the
vesicle. And if you remember Palade

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sequence, the next stop is the
Golgi. So the head over to the Golgi,

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they bind, they fuse, and what was
imbedded in the membrane is still

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imbedded in the membrane. And
the fully secreted protein is

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within the lumen of the Golgi.
Here the sugars are modified. I

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put little bows on them. And
they work there way over to the

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far side of the Golgi where
they bleb off again into

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secretory vesicles.