I was a graduate student in Arthur Kornberg's lab and I worked on
bacteria and got used to the advantages of working on a microorganism
that's genetically manipulable and can be grown in large quantities
and as a uniform population of cells.
And then when I became interested in biological membranes, I did a
postdoctoral period of study at UC San Diego with S.J.
Singer and worked on mammalian cells, specifically on human neonatal
erythrocytes, which were difficult to come by, and the technical
possibilities were very limited.
So I wanted to go back and work on a microorganism, but I wanted to
work on an organism that was more like human cells.
And yeast cells were -- had many of the advantages of bacteria, but
were quite distinct, and that had been made most clear by the work of
another Nobelist, Lee Hartwell, who used yeast genetics to study how
yeast cells divide by isolating mutations, lethal mutations, that
kill the cell but at different stages in their progression through
the division cycle.
So I was mightily influence by Hartwell, but also by Kornberg's style
of biochemistry.
So having experienced the difficulty of working with human specimens,
the limited availability of human cells, and at the time -- 1974, '75
-- the limited technical approaches that were available, I longed for
the advantages of a microorganism.
And when I returned to Berkeley, motivated by Lee Hartwell's work on
the cell division cycle using genetics to dissect the process of cell
division in yeast, I decided to adopt yeast as an experimental
organism, although I'd had no experience with yeast, nor had I done
genetics on anything.
But nonetheless I went to a three-week Cold Spring Harbor course
on yeast genetics.
I learned a little bit and I came to Berkeley and we started to look
at protein secretion, and after about a year we started to look
for mutations that blocked protein secretion.
The hope was that yeast cells would use a pathway similar to what
more complicated animal cells use, but there was no guarantee of that.
Nonetheless the genetic approach that Peter Novick and I developed
revealed that yeast cells have indeed the same organelles and
the flow of material in this pathway is the same as an animal cell's in
spite of a billion years of separation through evolution.
Well the very first mutant that we isolated we called sec1 --
short for secretion -- and it blocks the pathway at the very end.
Mature secretory capsules called vesicles build up throughout the
cell because they fail to migrate to the cell surface and discharge
their content by secretion to the cell exterior.
We didn't know what the gene was at the time. Indeed, when
we first isolated sec1, cloning of genes in yeast had
not technically advanced, and it took several more years until Gerald
Fink, then at Cornell, developed a technique to clone yeast genes.
In the meantime, we had repeated our procedures and isolated many
genes that when blocked -- when mutated, block traffic at different
stages in the pathway.
So we had been able to populate a pathway with mutations, but still
didn't have the genes.
So then it became possible to clone the genes and we and other labs
did this, and frustratingly, at first, the genes that we cloned
didn't look like anything; that is, they encoded proteins whose
function was completely obscure.
Around this time, the genomes of other higher organisms were being
sequenced and the corresponding genes, SEC genes from animals, indeed
from humans, became apparent and so we knew at least that these gene
products served some common function in all eukaryotic cells that
have a nucleus, even in nerve cells that secrete neurotransmitters.
And so with molecular tools, one could begin to see where these
proteins are located.
We now know through much work in many laboratories, including in
yeast, that the sec1 protein, that very first gene, encodes a protein
that is required for the organization of these vesicle carriers at a
target membrane.
It allows the membranes of the target and the vesicle to bridge
through the intervention of proteins that are called SNARE proteins
that are actually in the membrane and that this in sec1 then controls
the interaction between these SNARE proteins.
So a protein that in yeast simply blocks secretion and expansion of
the cell wall turns out to be absolutely crucial at the synapse to
organize the process of neurotransmitters secretion.
And that realization with that one protein -- and indeed with all of
the proteins -- that they serve these conserved functions throughout
the pathway, led to a real, I think, a deep mechanistic understanding
and appreciation of the conservation of this pathway.
Well, I'm a basic research scientist.
I've been guided by an abiding interest in how the cell works.
Mindful of the fact that when something fundamental is discovered, it
can have application, and so early on in my career I became an
advisor, a scientific advisor to a newly minted biotech company
called Kyron which formed in East Bay in the San Francisco area.
Their aspiration was to use yeast as a vehicle for the production of
medically useful proteins and they had me consult with them on how
they might harness yeast cells as a factory for human proteins.
Their first success was that they could take the gene that encodes a
protein in the hepatitis B virus and get that protein to be produced
in yeast, and they observed that the protein relied on membranes of
the secretory pathway to create little particles that were inside,
retained within the cell, and which resemble virus-like particles,
but which lack the infectious material normally associated with
hepatitis virus.
So they were able to express this protein in yeast, produce little
virus-like particles which were potent for immunization purposes, and
so that became the first large scale effective vaccine against
hepatitis B, indeed that's the world's supply of that vaccine is made
by expression of the hepatitis virus surface protein in yeast,
exploiting some aspect of the yeast secretory pathway.
Their next target was to again take advantage of the fact that yeast
secretes proteins by this pathway and to produce human insulin in yeast.
And I helped them as a consultant, and over a few years they were
able to engineer a copy of the human insulin gene and could produce
very large quantities of human insulin that folded properly and
functioned properly -- very different than the human recombinant
insulin form that Genentech had made by expressing the protein in
bacteria -- and that product was then taken over by a large
pharmaceutical firm in Europe, Novo Nordisk, and they now manufacture
one-third of the world's supply of recombinant human insulin by
secretion in yeast.
So although I didn't do those experiments in my lab, the fact that we
were able to show that yeast cells have a human-like pathway of
secretion emboldened the company to use yeast rather than
E. coli bacteria as a kind of a fermenter for the production of these
useful proteins, and so my feeling is that my best role was to
continue to inspect the mechanism of this process, doing basic
science in yeast, and to rely on the fact that the private sector is
free to exploit these, and it was my pleasure to help them do that
but it was not my intention to do that myself in my laboratory.
One has the conviction of the unity of biology and, you know, a
complicated process like secretion that involves, as we now know,
probably hundreds of different genes is not, you're not going
to reinvent the wheel every few millennia.
So the bits and pieces of that process would necessarily be
exploited; it simply remained to discover the mechanism, to discover
the molecules, and then to realize that they would -- to understand
that it was almost certainly going to be true in mammalian cells, but
you don't know until you do the experiments.
And the point was, really the motivating point was that it was
possible with technology available in 1976 to do this really simply
with simple tools like toothpicks and Petri plates, to do this in
yeast where it simply would not have been possible with that ease to
do it in human cells.
My first NIH grant was not funded.
It was proposed to work on this process in yeast.
The study section felt that I didn't have adequate experience.
I hadn't worked on yeast before.
They felt that I didn't have good preliminary results; of course
I didn't have any.
So my first grant was from the National Science Foundation.
They took a gamble on me and with that money I was able to, we were
able to isolate the first secretion mutants.
And then when I came back to the NIH three years -- or, two years --
after I started at Berkeley, suddenly they embraced me and they said,
"This is great. We'll give you money."
And they funded me generously for many years.
I was supported for, I don't know, over 25 years, but I let my
funding lapse because 22 years ago I became a Howard Hughes
investigator and their support has been very generous, and I felt
that I could sustain my research program with funds from Howard Hughes.
And I felt that, frankly, that I didn't need the extra money and that
it was better spent on people who actually needed the NIH support so
I've stopped applying for NIH support.
We've started a new project -- several new projects -- again, having
to do with membrane traffic largely in mammalian cells, and I talked
today in my seminar here at the NIH about a new project on how the
membrane that's involved in a process called autophagy forms.
Autophagy is a process whereby cells, when stressed by starvation,
envelope wholesale bits of cytoplasm and organelles and deliver these
sequestered proteins and organelles to the lysosome where they're
degraded, and where amino acids and sugars are then recycled for
reuse for the production of essential proteins that would allow a
starved cell to survive.
So it's a very important process of cellular renewal under conditions of starvation.
And there's been an open question about where this membrane that
forms this envelope that collects these organelles, where it forms
and how it grows, and I have a wonderful postdoctoral fellow who's
developed a cell-free reaction that reproduces an early event in this process.
And we think we now have biochemical access to the entire pathway
using the tools of the biochemist and enzymologist.
So I'm very excited about this.
And, you know, we're off and running on that project now.
So, tumor cells seem to be particularly dependent on the autophagic
process, for reasons that I'm not sure are entirely clear, and so
drug trials have been conducted for inhibitors of autophagy that seem
to enhance cell death by interfering with this process.
So tumor cells may be somehow more dependent than normal cells on
this turnover process to replenish themselves to provide nutrients
for ongoing growth and division.
So it's promising, then, to be able to interfere with that and maybe
to have some chemical treatment for those tumors that are dependent
on this pathway.
Again, my interest is in how the cell basically organizes this and
the details of that.
And again I feel it's better left to the private sector to try to
exploit what knowledge we gain to intervene and develop therapies.
Well, as I said, what we've developed is a new way of studying this
process that's not limited looking at intact cells.
We can now break the cell open and reproduce a significant chunk of
this pathway in the test tube.
And that means that we can manipulate the system much more easily
than one can in an intact cell -- you can purify the enzymes that are
involved, you can eventually put them back together in pure form with
an isolated membrane and study in some detail exactly how the pathway works.
This is what we've done in studying the secretory pathway.
For me, it wasn't satisfying to isolate the mutants or even to clone
the genes, because as I said, the genes, at least initially, weren't
terribly instructive.
So to me, and again from my graduate training, I felt it would be
necessary to reproduce in the test tube these events and then be able
to purify the relevant molecules and study them in isolation.
Indeed, in that case we really were able to make discoveries using
this biochemical approach that neither the genetics nor the molecular
biology would have offered us.
And I am equally confident that this new approach in studying
autophagy will be instructive in that way.
Well, since I'm here at the NIH and this is the first lecture,
public lecture I've given since I won the Nobel prize, I want to
express my gratitude, not only to the NIH for the generous support
they provided over the years; to the government for the wisdom of its
investment in basic science; and, very specifically in my case, to
the people of California, who have invested wisely in creating this
great university system, the University of California, where I was
trained as an undergraduate, as a postdoctoral fellow, and now for
the past... over 37 years as a faculty member.
Without that wise investment of public funds and basic science and
then public higher education, my career would have gone in a very
different direction.