Hello, I am Peter Walter. I work at the University of California at San Francisco,
and we are basically cell biologists trying to figure out how cells work,
specializing on organelle biogenesis and how proteins
get to the right place and how they function.
And ultimately we would like to understand how proteins work as molecular machines
to make cells as wonderful and as complicated as they are.
So what I want to tell you today is a brief story of discovery
I think one of the most wonderful discoveries that was made in my lab in my career,
and it involves the reaction by which cells
decide how much of a particular organelle they have to have.
And the organelle that I'm going to tell you about is the endoplasmic reticulum,
which is the first way station which proteins enter
as they move through the secretory pathway to the surface of the cell,
and end up being secreted or inserted in the plasma membrane.
The endoplasmic reticulum then is a place where proteins have to...
They enter the ER; they have to fold;
they have to become assembled from multiple subunits; they become modified,
maybe a carbohydrate is added; disulfide bonds are formed.
And all of these reactions are important to produce properly functioning proteins.
And cells that are specialized, that make a lot of proteins and secrete them,
have to have a lot of endoplasmic reticulum
in order to carry out these processes with fidelity and with appropriately abundant machinery.
The proteins enter the endoplasmic reticulum
in an unfolded state as they come out of the ribosome
and then as they are in this organelle they basically have to mature.
And if they cannot fold properly,
then a cell basically puts proteins in its plasma membrane or secretes them
and because all the machinery that is required for a cell to know where it is in the body,
what it has to do, how it has to behave in the context of a multicellular organism,
all this information is being transmitted by proteins that are being secreted
or by the machineries that sit in the plasma membrane.
So it's very, very important that these machineries work properly, are properly assembled
because otherwise you may create a rogue cell that doesn't know when to divide,
when to differentiate, when to die, when to migrate to another place and so on and so forth.
So, for the organism, this is a very important process.
And this is sort of amplified in this slide
where you look at the differentiation of this cell, a precursor cell here
that turns into a professional secretory cell, in this case it's a plasma cell.
And the plasma cells are the cells that make antibodies that are being secreted in the bloodstream,
so this cell makes its own weight every day in antibody molecules.
And you see that this differentiation event goes hand in hand
with this vast proliferation of endoplasmic reticulum
that basically fills the cytosol wall to wall.
So, how does that cell know how much endoplasmic reticulum it needs?
So there must be signaling pathways in this cell that figure out how much of an organelle
we have and how much should be there according to the needs of the secretory load that the cell has.
And this signaling pathway that transmits that information,
which we've been studying for the last fifteen years or so,
is called the Unfolded Protein Response.
And it's called the Unfolded Protein Response because it initiates with an accumulation of
unfolded or misfolded proteins in the endoplasmic reticulum.
And these proteins, they are unfolded because there isn't enough capacity
to fold them properly from the machinery.
These proteins then create a signal that's being transmitted
across the membrane of the endoplasmic reticulum
that ends up eventually in the nuclear compartment
where it turns up a vast gene expression program
that makes, basically, more endoplasmic reticulum folding capacity, secretory capacity,
capacity to degrade proteins that cannot be folded properly and so on and so forth.
So it brings cells back into a homeostatic state that allows the load of secretory proteins
to be balanced with the machinery available to carry out their task.
To figure out how this pathway works two very adventurous graduate students in my lab,
Jeff Cox and Carolyn Shamu, started a project in which we tried to identify
these components genetically in the yeast system.
And basically, what they did is they built a reporter system based on an observation
made by Kazutoshi Mori in Mary-Jane Gething and Joe Sambrook's lab
that there is a small element in the promoters of the target genes of the response
that can be transplanted, can be put in front of a reporter gene,
and then we can isolate mutants in the cell where when we induce unfolded proteins in the ER
that no longer induce the Unfolded Protein Response by this induction of this reporter gene.
We can then take these mutants, we can clone the genes that have been mutated
by complementation and figure out what they do in the pathway.
And the nice thing is that the first gene, which we isolated this way, turns out
to be IRE1 and it encodes a transmembrane kinase.
So by virtue of it being a transmembrane kinase,
it already told us that this may be the signal transduction device
sitting in the ER membrane, figuring out in one end what's going on there,
and transmitting that information across the bilayer.
Very nicely, the second gene we isolated turns out to be HAC1,
and HAC1 encodes a transcription factor that binds to all these promoter elements.
So we then have the transmembrane kinase, we have a transcription factor,
and of course the way we are thinking about that
is very much in analogy to other transmembrane kinases,
like growth factor receptors in the plasma membrane of mammalian cells,
that this thing is activated and functions
by a process of oligomerization in the plane of the membrane
where as unfolded proteins accumulate, these kinase molecules come together,
they start bringing the kinases together on the other side of the membrane
where they are now juxtaposed so they can trans-autophosphorylate each other
and that somehow leads to phosphorylation cascade
downstream that activates the transcription factor.
But it turns out that nothing could be further from the truth.
This pathway is wired in a completely different way and a completely unexpected way.
And that was discovered, pretty much, by a series of control experiments that Jeff carried out.
And he was the first one, very simply, very naively,
just started looking for the transcription factor in cells
that are either induced for the response or that are not.
And as you can see here, the transcription factor is only present
in cells when the response is induced.
So that tells us two things right then and there, right?
It's either degraded when its not needed
or it is only synthesized when the response is induced.
And to distinguish between these two possibilities what Jeff did is
he just did a simple Northern blot analysis by which he asked,
Does the messenger RNA encoding this transcription factor
change in its abundance when we induce unfolded proteins?
As you see here, the messenger RNA doesn't really change much in abundance.
But what you see, what Jeff discovered here, is that we now have a band of a different size
that was completely unexpected. So, the simple Northern blot then led to the discovery
that there is something happening to the messenger RNA and to make a long story short
it turns out that this messenger RNA becomes spliced.
An intron is being removed when the Unfolded Protein Response is induced.
So the idea then is, that the messenger RNA encoding
the transcription factor is initially encoded with this intron
and as the unfolded proteins accumulate, this intron is being removed,
producing the spliced messenger RNA which is then being translated
to produce the transcription factor that turns up the response.
Now this is highly unusual. Normally, when cells decide to change their transcription
to their splicing program through some alternative splicing,
they make reasonably irreversible developmental decisions
so that they last a long time. They enter different cell fate with these decisions.
But this is bona fide signaling.
What's happening here is...strictly dependent on
the conditions inside the endoplasmic reticulum.
The signaling is on and off depending on whether you have unfolded proteins or not.
What's even more surprising is that this splicing reaction
followed none of the rules of normal messenger RNA splicing.
It's completely independent of the spliceosome. It's happening in the cytosol and
it's carried out by two enzymes and two enzymes only.
The first one turns out to be Ire1.
Our transmembrane kinase when activated, becomes a bifunctional protein that is not only a kinase,
but also a site specific endoribonuclease that cleaves the messenger RNA
encoding the transcription factor precisely at both splice junctions
and to the best of our knowledge, this is the only messenger RNA in the cell that it touches.
And then, along comes an enzyme called tRNA ligase
which was previously only known for its role in tRNA splicing
and we had isolated a mutation in that, another graduate student, Carmela Sidrauski,
and we couldn't make any sense out of that until Jeff had discovered
that there's this RNA processing step in the signaling pathway.
So the Unfolded Protein Response then transmits the signal by this unconventional,
completely unprecedented splicing pathway
and there's no phosphorylation cascade anywhere in sight here.
Let me just show you this, too. We can make this messenger RNA in vitro,
we can incubate it with recombinantly produced Ire1
and you see that the messenger RNA gets cleaved,
producing the intron, 5' exon, 3' exon and then as we add also recombinantly produced,
purified tRNA ligase to this reaction the exons go away,
get ligated to form the product and the intron stays put.
So we can reconstitute this whole pathway
from two purified components with quite nice efficiency.
To make a long story short, this discovery then led to many other labs and it turned out that
pretty much everything...the cellular features that we've learned from
the simple yeast system hold true for mammalian and metazoan cells.
So this IRE1 exists in metazoan cells, it is involved in the splicing of a messenger RNA
encoding a transcription factor, XBP1, XPB1 here.
Things are more complicated, higher evolved eukaryotic cells have
added more bells and whistles to the pathway.
So we have three parallel pathways here that transmits information from the ER lumen
to the cytosol each leading to the activation of a transcription factor.
We have Ire1 working by this non-conventional mRNA splicing.
We have another transmembrane kinase here, PERK
that makes another transcription factor by a mechanism of translational control.
And finally, we have this exciting protein here
that sits in the ER membrane, ATF6, and then gets released,
a fragment of it gets released to become an active transcription factor
that moves into the nucleus and turns up the target genes only when
unfolded proteins are accumulating in the endoplasmic reticulum.
And the idea of the whole thing is the same as I told you before.
It is to establish, re-establish homeostasis so protein folding
in the endoplasmic reticulum can occur with fidelity.
So we have these three pathways here and they establish homeostasis.
But I also told you that there is a danger that if cells cannot achieve homeostasis
that they may make mistakes in protein folding and therefore turn into rogue cells
that endanger an organism. So there's a safety valve built in
that if this balance cannot be achieved again that cells, rather than
putting these misfolded proteins, and misfolded signaling machines on the cell surface
go down a pathway of apoptosis.
So rather than becoming rogue cells and endangering the organism,
they remove themselves by committing suicide.
And it's this point that the Unfolded Protein Response makes
life death decisions for the cell that puts this pathway in the midst of many different human diseases.
Some cancer cells are kept alive because the response gives them a growth advantage.
In diabetes, the beta cells in the pancreas may die through this apoptotic route here
by being over-committed to produce ever increasing amounts of insulin.
And there are neurodegenerative diseases where protein misfolding causes apoptosis
by turning up this response.
So, basically then, our very pioneering work of these very adventurous graduate students
has led us to understand a mechanism that we now, we and many other labs
have tried to utilize in therapeutic approaches to see if we can have
some ability of bettering mankind via interfering and modulating these pathways
and therefore affecting the outcome of human disease.
And I should close by giving credit to these really wonderful, adventurous pioneers:
Jeff Cox, who discovered, together with Carolyn Shamu, Ire1 and the Hac1 transcription factor
and Carmela Sidrauski, who discovered that Ire1 is a nuclease in the splicing reaction and
put the tRNA ligase on the map.