Uncovering the Secretory Pathway

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00:00:13.22 Hello, I am Ron Vale.
00:00:16.06 Today I have the great
00:00:16.23 pleasure to be speaking with
00:00:18.02 Jim Rothman and Randy Schekman,
00:00:20.09 who just returned
00:00:21.00 to the United States from Stockholm
00:00:23.03 where they received the 2013
00:00:25.13 Nobel Prize in Physiology or Medicine
00:00:27.29 along with their colleague Tom Sudhof.
00:00:30.21 This trio was awarded the prize
00:00:32.28 for their seminal work in uncovering
00:00:36.04 the molecular machinery that is involved
00:00:38.10 in membrane trafficking
00:00:40.08 and protein or neurotransmitter secretion.
00:00:43.14 This is a journey of proteins
00:00:45.08 through a cell
00:00:46.11 that starts with protein insertion
00:00:48.21 into the endoplasmic reticulum.
00:00:50.09 Those proteins are then recognized
00:00:54.08 as cargo, packaged,
00:00:55.22 and then transported
00:00:57.24 as small vesicles
00:00:58.23 to the next way station,
00:01:00.07 which is the Golgi apparatus.
00:01:01.24 And from there
00:01:03.14 they are transported to new
00:01:05.17 membrane destinations,
00:01:06.18 such as the lysosome,
00:01:07.25 or the plasma membrane.
00:01:09.12 And this trio of Sudhof, Rothman, and
00:01:12.25 Schekman found the
00:01:14.29 molecular machinery
00:01:16.03 that is involved in each step
00:01:17.24 of this pathway through the cell.
00:01:20.03 So thank you for joining us today.
00:01:24.01 And I'd like to begin
00:01:26.13 with the science.
00:01:27.11 And maybe, Randy, I could ask you
00:01:29.20 if you could describe the work
00:01:32.22 that Jim did that led to his
00:01:35.20 contributions for the Nobel Prize.
00:01:37.13 Schekman: I am happy to do that.
00:01:38.24 I first met Jim when he interviewed
00:01:41.11 for a faculty position at Berkeley.
00:01:43.05 As an assistant professor I had been there
00:01:45.25 about a year.
00:01:47.23 And he told me in broad
00:01:49.10 terms about his plan,
00:01:51.06 which I certainly
00:01:52.21 appreciated being a biochemist.
00:01:54.15 And then he went and did it at Stanford.
00:01:57.16 Very quickly, he launched
00:02:00.09 into developing a cell-free reaction
00:02:03.18 to reproduce traffic, at least initially,
00:02:06.19 he thought it was traffic
00:02:07.22 from the ER to the Golgi.
00:02:08.27 Subsequently he turned out to be
00:02:11.14 measuring traffic,
00:02:13.00 vesicular traffic, within the Golgi.
00:02:14.23 And he used that to identify
00:02:18.01 soluble proteins
00:02:19.19 that he called NSF, for N-ethylmaleimide
00:02:22.13 sensitive protein
00:02:23.18 and its SNAP, which
00:02:25.10 was its membrane receptor for NSF,
00:02:29.04 and showed that these
00:02:31.20 were involved at some point
00:02:32.19 in membrane fusion.
00:02:34.17 And then in a really brilliant experiment,
00:02:37.04 several years later,
00:02:38.04 he used the
00:02:39.03 NSF and alpha-SNAP complex,
00:02:41.20 bound by non-hydrolyzable ATP analog,
00:02:45.03 to fish out of membranes
00:02:47.19 from a crude detergent soluble fraction
00:02:50.07 from bovine brain
00:02:51.28 the proteins that turned out
00:02:54.05 to be the membrane anchors
00:02:57.00 and the catalysts for fusion,
00:02:59.06 which he called SNARE proteins.
00:02:59.26 So it was a really beautiful synthesis
00:03:02.11 of the biochemistry
00:03:03.28 and functional analysis.
00:03:06.22 Vale: And Jim maybe
00:03:07.28 I could ask you the same question.
00:03:09.25 Could you comment
00:03:10.20 on the key discoveries and contributions
00:03:13.02 that Randy made
00:03:14.03 that led to his
00:03:15.12 being awarded the Nobel Prize?
00:03:17.01 Rothman: This is a great pleasure
00:03:19.06 to have a chance to.
00:03:21.08 Randy and I have been joking
00:03:22.07 for a long time
00:03:22.23 about giving each other's seminars,
00:03:24.09 so you've given us
00:03:25.07 a great chance to do that, Ron.
00:03:26.00 Randy in the... starting in the late 1970s,
00:03:33.01 took on a problem
00:03:35.06 that was extremely adventurous.
00:03:38.16 And people at that time,
00:03:39.17 model organisms were not
00:03:41.18 what they are today.
00:03:42.16 There were very few examples
00:03:45.00 where for example yeast had been used
00:03:47.07 to really forge ahead and understand
00:03:51.06 complex eukaryotic pathways.
00:03:52.09 As Randy points out,
00:03:54.23 the yeast cell cycle was one.
00:03:57.18 People doubted actually
00:03:58.27 that there was a secretory pathway in yeast
00:04:00.18 that reflected in any meaningful way
00:04:06.21 the similarity to the Palade pathway
00:04:09.23 that we all had understood at the time.
00:04:12.02 The Golgi apparatus, if it was observed,
00:04:15.06 it was only scantily observed.
00:04:17.16 And Randy especially
00:04:20.22 in a really innovative approach
00:04:22.19 was able to isolate a series of mutations.
00:04:24.26 First based on kind of brute screening,
00:04:28.23 and then after that based on a
00:04:30.18 really important experimental insight
00:04:33.26 about if you have a cell
00:04:35.13 that is making protein
00:04:36.22 but not excreting the protein
00:04:37.19 it's going to get denser.
00:04:39.25 So based on that
00:04:40.26 Randy was able to isolate
00:04:42.18 a large number of mutations
00:04:44.00 systematically that essentially defined
00:04:45.26 the entire secretory pathway
00:04:47.00 and the major secretory organelles in yeast.
00:04:50.01 And established the generality
00:04:52.09 of the pathway. But more importantly
00:04:53.17 they provided the means to discover
00:04:56.01 the molecular machinery
00:04:57.20 that is important in various stages
00:05:01.08 of vesicle budding and fusion.
00:05:02.15 And it's turned out
00:05:03.09 that over the years that
00:05:05.09 the genes that Randy
00:05:06.16 initially discovered,
00:05:07.18 23 of them,
00:05:08.04 play pivotal roles in multiple stages
00:05:11.17 of the secretory pathway.
00:05:12.10 Some at all stages of vesicular trafficking.
00:05:15.13 His students and others
00:05:19.00 have followed in his footsteps
00:05:20.21 and have essentially
00:05:21.09 laid out the entire map
00:05:25.00 of the transport pathways
00:05:26.06 in the yeast cell.
00:05:27.01 Vale: So both of you
00:05:28.18 shared some interesting similarities
00:05:30.17 in terms of how your careers started.
00:05:33.18 You're both trained as
00:05:35.08 biochemists in some great laboratories.
00:05:38.04 And you then became assistant professors
00:05:42.13 both at a very young age.
00:05:44.04 And then both of you picked
00:05:47.13 very risky problems to start your laboratories.
00:05:50.03 So I'd like to get some background
00:05:52.03 of how you came up
00:05:53.11 with this project,
00:05:54.04 and how you decided to tackle it.
00:05:57.05 So maybe I could start with you Randy?
00:05:59.09 Maybe you can just tell me how you ended up
00:06:03.02 choosing the problem membrane secretion
00:06:04.24 and also taking a genetic approach.
00:06:07.08 Schekman: Near the end of my graduate career,
00:06:09.01 in looking at the literature on membranes,
00:06:11.26 largely influenced by Bill Wickner
00:06:14.08 who was a postdoc in Arthur's lab
00:06:15.22 at the time who also had come from Kennedy's lab
00:06:18.00 that biological membranes were likely to be
00:06:24.26 a subject that one could
00:06:28.01 learn a great deal about biosynthesis.
00:06:30.17 One could possibly treat
00:06:32.09 a biological membrane
00:06:33.22 as a macromolecule
00:06:34.25 and dissect its biogenesis
00:06:36.12 using the techniques of
00:06:38.00 biochemistry and genetics
00:06:39.07 in a way that hadn't really
00:06:40.26 been applied yet.
00:06:42.28 So I went to John Singer's lab
00:06:45.10 to learn about what he was thinking
00:06:47.09 about biological membranes
00:06:48.15 and I found myself on a project
00:06:50.24 that didn't really have a future in my mind.
00:06:54.16 It was using a scarce material,
00:06:57.24 and I found that
00:06:58.20 working with mammalian cells,
00:07:00.17 at least at that time
00:07:00.25 in the mid 1970s
00:07:02.10 was not as satisfying as working with a microorganism.
00:07:06.03 So I resolved when I joined
00:07:08.17 the faculty at Berkeley
00:07:09.21 influenced to some extent,
00:07:11.25 some large extent
00:07:13.15 by the success of Lee Hartwell
00:07:14.11 in his genetic dissection
00:07:16.03 of the cell division cycle
00:07:17.06 that I would then
00:07:18.05 launch an independent career using
00:07:20.15 some kind of genetic analysis
00:07:22.09 of membrane biogenesis.
00:07:26.07 And Peter Novick
00:07:27.12 was one of my first graduate students
00:07:30.02 and it was my good fortune that he joined my lab.
00:07:33.09 He was a very thoughtful, very precise,
00:07:35.22 hardworking, really brilliant student.
00:07:40.08 And so we, the two of us,
00:07:41.23 sort of embarked on this
00:07:42.21 effort to identify
00:07:44.14 the genes involved in secretion
00:07:46.26 by a genetic approach.
00:07:47.24 It was kind of a trial and error.
00:07:49.28 Initially we had some crazy idea
00:07:51.14 about the need for a genetic selection
00:07:53.18 that didn't work.
00:07:54.18 We found a mutant in spite
00:07:55.12 of it being a selection that didn't work
00:07:57.12 we found the first mutant anyways,
00:07:59.11 and we realized that it was a complex pathway
00:08:02.16 and that probably just looking
00:08:03.27 through a random assortment
00:08:05.03 of temperature sensitive mutants,
00:08:06.19 just as Hartwell had done,
00:08:07.19 would turn up more genes.
00:08:09.24 And we did find another one,
00:08:12.20 but as Jim mentioned earlier,
00:08:15.02 we decided to use an enrichment procedure
00:08:18.10 and isolated a lot more mutations.
00:08:20.04 So it was a good choice obviously.
00:08:24.28 It worked out well.
00:08:25.28 It's not the traditional way that
00:08:28.21 one starts a career.
00:08:30.01 Usually postdocs are
00:08:31.17 a little more conservative
00:08:33.07 but I always advise my postdocs
00:08:35.18 to be more bold
00:08:36.26 and to do something new.
00:08:38.10 But of course when you do something
00:08:40.14 new there's a good chance you will fail.
00:08:42.02 So what I did was I started
00:08:43.27 several different projects.
00:08:45.02 This one worked within a year.
00:08:46.29 And then I marshaled
00:08:47.28 all of my efforts to focus on that.
00:08:49.09 Vale: So as a follow up, I was wondering
00:08:51.23 if you could explain the assay that you used
00:08:54.13 to get the second mutant.
00:08:56.28 And also your reaction
00:08:58.04 to that amazing electron micrograph
00:09:00.21 in the Figure 6 of your 1979 paper
00:09:03.20 that shows this yeast cell
00:09:05.28 filled with all of these vesicles
00:09:08.24 that could no longer reach the cell surface.
00:09:10.18 Schekman: So the idea was that in looking at
00:09:14.04 thin sections in yeast cells
00:09:15.28 one can see some organelles
00:09:18.25 that look like they could be associated
00:09:20.22 with secretion.
00:09:21.13 Not as dramatic as in a section
00:09:23.00 of mammalian cells, but Novick and I guessed
00:09:25.28 that the vesicles that one sees
00:09:27.21 congregating under the bud portion of a dividing cell
00:09:29.29 would be the vehicle not only for secretion,
00:09:33.07 but for assembly of the plasma membrane.
00:09:34.08 And so we predicted
00:09:35.18 that anything that blocked
00:09:37.11 the biogenesis of these vesicles
00:09:38.21 would be lethal
00:09:39.25 and that therefore that one would have
00:09:41.20 to go after conditional,
00:09:42.22 temperature sensitive
00:09:43.24 lethal mutations.
00:09:44.24 So that was our first and crucial assumption
00:09:49.04 that it was going to be an essential gene.
00:09:51.11 And the second was that
00:09:53.02 the mutant would,
00:09:54.16 at a restrictive temperature,
00:09:56.09 cause secretory proteins,
00:09:57.18 which are otherwise very efficiently
00:09:59.13 secreted from the cell,
00:10:00.20 to accumulate inside the cell.
00:10:04.08 And that actually was a really trivial assay.
00:10:06.18 You just assay intact cells,
00:10:08.11 which measures the enzyme
00:10:10.00 that has been trapped in the cell walls.
00:10:11.14 The cell wall is porous.
00:10:12.28 And then you dissolve the cell wall
00:10:14.07 with an enzyme,
00:10:15.25 and you have spheroblasts left.
00:10:17.17 And then you just rupture
00:10:18.16 the spheroblasts with detergent
00:10:19.28 and you assay what is
00:10:20.21 inside the spheroblast,
00:10:23.01 and that is a measure of
00:10:23.18 the accumulation.
00:10:24.11 In a wildtype cell,
00:10:25.13 that baseline is virtually zero.
00:10:28.10 In a sec mutant it rises dramatically
00:10:30.25 above the background.
00:10:32.09 So the first mutant,
00:10:33.00 just based on a biochemical
00:10:34.25 enzyme assay was really obviously
00:10:37.24 blocked in multiple
00:10:39.03 secretory enzyme transport.
00:10:41.12 But the dramatic result came
00:10:43.01 just around the time
00:10:44.05 that George Palade visited Berkeley
00:10:46.09 for a special lecture
00:10:47.23 and I recall Novick telling me
00:10:49.23 at the luncheon that
00:10:51.01 Palade had encouraged him
00:10:52.04 with these preliminary results
00:10:53.12 to cut a thin section
00:10:54.09 and look and see what these cells looked like.
00:10:55.27 And so we got an expert technical assistant
00:10:59.09 in the department,
00:11:00.05 and that of course was one of the
00:11:01.29 great eureka moments of my career
00:11:03.22 when Novick excitedly
00:11:05.16 called me down to the EM room
00:11:09.12 and showed me these
00:11:09.17 sec1 cell sections
00:11:11.03 that were like they had measles.
00:11:13.21 They were just full of vesicles
00:11:14.21 and nothing like that
00:11:16.16 had ever been published.
00:11:18.12 I don't think it had ever been
00:11:19.06 seen before.
00:11:20.16 And from that moment,
00:11:21.11 literally from that moment
00:11:23.01 on, I knew we would have at least
00:11:25.09 several decades worth of productive work.
00:11:28.19 Vale: Well Jim, let me ask you
00:11:30.10 a similar question.
00:11:33.05 You started off your career at Stanford
00:11:34.12 as a very young assistant professor
00:11:37.13 You had experience with the VSV virus from
00:11:42.04 your previous work,
00:11:43.26 but you decided to tackle
00:11:46.02 a very difficult problem,
00:11:48.11 which was to try to reconstitute
00:11:49.19 parts of the protein secretion
00:11:53.10 pathway in a cell free extract.
00:11:56.28 And in fact many people
00:11:59.05 thought that wouldn't work.
00:12:00.05 And that it would be
00:12:01.04 an extremely risky project to take
00:12:02.24 on so I was wondering if you
00:12:04.02 could give a little bit about
00:12:06.21 your thinking
00:12:08.21 and the background that
00:12:08.26 led to that particular project.
00:12:10.27 Rothman: My exposure to
00:12:13.05 this entire area of
00:12:15.05 biology began as a medical
00:12:16.10 student in my first year
00:12:17.22 at Harvard Medical School.
00:12:19.01 And George Palade's
00:12:21.17 discoveries were really fresh
00:12:24.18 and they were presented
00:12:24.28 in my histology course.
00:12:27.01 And when I saw those
00:12:29.00 electron micrographs of vesicles
00:12:30.20 and endoplasmic reticulum
00:12:31.24 and Golgi
00:12:32.09 I immediately started
00:12:34.14 thinking given my background
00:12:36.12 in physical sciences,
00:12:39.11 how in the world can such a vesicle from, you know...
00:12:40.27 what would make that happen,
00:12:41.26 and how would it know where to fuse?
00:12:43.25 And I must say somehow I identified
00:12:46.08 that problem as one that
00:12:47.17 I wanted to solve
00:12:48.09 at some point and I got fixated on it.
00:12:51.20 And I can't really tell you why,
00:12:53.05 but I just got fixated on it.
00:12:54.15 I found it as fascinating then as I do now.
00:12:57.04 And after leaving medical school
00:13:02.07 and finishing my PhD with Gene Kennedy,
00:13:04.25 I did learn some basic biochemistry
00:13:06.19 of membranes and lipids.
00:13:08.22 I went to work with Harvey Lodish
00:13:09.21 for a year or two,
00:13:12.08 and I did it with the freedom
00:13:13.17 of having already my assistant professor
00:13:16.02 offer in hand.
00:13:17.18 So I was able to do pretty much what
00:13:19.03 I wanted and focus
00:13:19.22 on really educating myself.
00:13:23.03 And from Harvey and his colleagues
00:13:24.14 I learned how to work with
00:13:27.04 complex cell free systems
00:13:28.07 where we were using cell free extracts
00:13:29.21 that were synthesizing protein.
00:13:32.24 We learned following the footsteps
00:13:34.03 of Gunter Blobel how to
00:13:36.27 study the incorporation of those proteins
00:13:38.23 in vitro into the endoplasmic reticulum
00:13:40.29 membrane and that
00:13:42.12 really equipped me very well.
00:13:43.09 Now when I went to Stanford
00:13:44.13 in 1978, like Randy
00:13:48.11 I pursued several different projects.
00:13:50.03 And that was a way of distributing the risk,
00:13:53.16 but then again I made a
00:13:53.24 decision to focus intensively
00:13:56.01 on one project when
00:13:57.24 we did have some success
00:13:59.09 after some period of time,
00:14:00.21 and that is the project
00:14:01.16 you're referring to
00:14:02.11 the reconstitution of
00:14:04.19 transport in a cell free extract.
00:14:06.00 And I think that decision
00:14:07.04 that Randy made
00:14:08.02 and the decision that I made
00:14:09.05 is a very important
00:14:10.13 decision that young scientists
00:14:12.01 can make which is
00:14:13.05 that at some point you really have to focus.
00:14:15.12 And you have to focus deeply on something.
00:14:16.27 And in the long run if you want to make
00:14:18.16 a contribution that is really worth making
00:14:20.23 rather than simply incremental,
00:14:22.20 you have to stick
00:14:24.06 as Randy did and
00:14:24.29 frankly as you did Ron
00:14:26.18 to that decision.
00:14:29.10 It's not always going to be easy.
00:14:31.03 And it's certainly not free of risk,
00:14:32.13 but it has great rewards
00:14:34.16 for science when you are successful.
00:14:36.06 So what we did was we
00:14:37.15 wanted to reconstitute transport
00:14:39.29 from the ER to the Golgi
00:14:41.04 so we took the cells,
00:14:41.23 living, we gave them a
00:14:43.23 very short pulse label
00:14:44.21 with radioactive methionine,
00:14:46.19 and tricks I had learned in Harvey Lodish's lab,
00:14:49.27 then homogenized
00:14:51.05 the cells to retain them,
00:14:54.13 the G protein, in the endoplasmic reticulum.
00:14:56.10 And then we incubated that extract
00:14:58.11 to see if the glycoprotein
00:15:00.08 could be further processed
00:15:01.18 by adding certain sugar residues,
00:15:05.06 especially N-acetylglucosamine,
00:15:05.25 which we knew
00:15:06.20 from the work of Phil Robbins
00:15:08.08 and Stuart Kornfeld
00:15:09.12 and others occurred in the Golgi.
00:15:13.04 And we could see that processing in vitro,
00:15:14.24 it would have suggested
00:15:15.21 the transport occurred.
00:15:18.14 And indeed, after I don't know,
00:15:20.05 a month or two,
00:15:21.27 we observed
00:15:22.14 that that was the case.
00:15:23.28 We could see it.
00:15:25.01 But we had a huge background.
00:15:26.21 Even without any kind of incubation
00:15:30.18 longer than a few seconds,
00:15:31.29 even without adding ATP or cytosol
00:15:34.20 we still saw processing and what we
00:15:37.06 came to realize was that
00:15:38.14 we already had too much of the G protein
00:15:39.23 sitting in the Golgi
00:15:40.26 and it was getting glycosylated
00:15:43.03 directly there. So that gave me the idea
00:15:45.09 of what is now known as a complementation assay
00:15:48.12 which is pretty much
00:15:49.11 the standard design for most
00:15:50.19 cell free (in those days)
00:15:52.15 cell free reconstitution of transport
00:15:54.29 and the idea is you want
00:15:56.26 to mix two extracts,
00:15:57.29 one from the cell that contains the cargo protein, VSV-G,
00:16:00.19 but lacks the processing enzyme,
00:16:03.00 The other extract
00:16:04.14 from the cell that
00:16:06.13 lacks the processing enzyme,
00:16:08.14 but... excuse me.
00:16:10.00 Has the processing enzyme,
00:16:10.29 but lacks the cargo protein.
00:16:13.09 And so when the two are mixed,
00:16:15.00 if the cargo protein
00:16:16.19 is getting glycosylated,
00:16:18.19 it must have gone from
00:16:19.28 a donor membrane
00:16:21.03 in the extract of the one cell
00:16:24.13 to the processing enzyme
00:16:25.20 in the Golgi
00:16:26.02 of the other cell.
00:16:27.29 And so that eliminated the background
00:16:29.17 that I referred to
00:16:30.25 because the extract
00:16:32.19 with the VSV-G protein in it was
00:16:34.10 incapable of processing by adding sugar.
00:16:37.21 So when we set that assay
00:16:38.28 up it actually worked.
00:16:40.14 It was in I believe 1979.
00:16:43.08 I still have the gel
00:16:44.10 that shows a very, very faint band
00:16:46.00 of the VSV-G protein being
00:16:47.15 processed to what is called an
00:16:50.01 endo-H resistant form.
00:16:51.10 And it was very, very exciting.
00:16:53.20 Vale: Randy, you were
00:16:55.22 originally trained as a biochemist
00:16:57.18 with one of the master
00:16:58.12 biochemists, Arthur Kornberg.
00:17:00.02 You then began to
00:17:02.14 pursue this problem
00:17:03.20 of membrane secretion in yeast
00:17:05.06 using a genetic approach.
00:17:09.04 When did you decide it was the right moment
00:17:10.16 to start to begin a
00:17:12.08 biochemical dissection of this problem?
00:17:13.29 Schekman: Our approach to initiate
00:17:16.18 with genetics was always, in my mind,
00:17:20.06 just a stepping-stone into biochemistry.
00:17:25.09 It as not going to... I didn't want to pursue it
00:17:27.27 purely by genetic analysis.
00:17:30.16 I had every intention of using
00:17:32.22 this to develop some
00:17:34.02 kind of cell-free reaction.
00:17:35.16 It was a challenge to get people
00:17:37.24 in my lab to think that way,
00:17:39.17 because when something is working
00:17:41.15 everybody who joins your lab
00:17:43.01 wants to keep doing that thing.
00:17:44.19 They wanted to isolate more mutants.
00:17:45.26 They wanted to clone more genes,
00:17:48.04 but convincing any of them
00:17:49.16 to actually take a step back
00:17:50.17 and do something very different,
00:17:53.01 which to me would have been
00:17:54.22 the obvious thing
00:17:55.24 was tough.
00:17:57.17 So fortunately after six or seven years,
00:18:02.19 David Baker joined the lab.
00:18:03.25 And he was brash
00:18:05.16 and considered our efforts
00:18:08.00 in that direction
00:18:08.25 inefficient and ineffective.
00:18:11.13 And within two weeks
00:18:12.20 he had developed a cell free reaction
00:18:15.01 that reproduced the first half
00:18:16.18 of the secretory pathway in vitro.
00:18:19.24 And from that point on,
00:18:20.28 everyone that joined the lab said okay,
00:18:22.24 I want to do the biochemistry
00:18:24.02 because it was obvious
00:18:24.21 how valuable that would be.
00:18:26.14 So what's the lesson here?
00:18:28.23 I guess you have to have
00:18:29.25 the courage of your own convictions
00:18:31.15 and be persistent
00:18:34.07 and I think particularly this kind of
00:18:34.21 biochemical approach if you had
00:18:37.17 success in that in the past
00:18:38.20 you have as an article of faith,
00:18:40.15 really certainty,
00:18:43.03 that you can do it again.
00:18:44.00 And I felt confident that we could
00:18:46.22 and of course, Jim had succeeded,
00:18:48.10 so I felt doubly confident
00:18:49.12 that we could do it.
00:18:50.02 And that was a plan
00:18:53.07 right from the start.
00:18:54.12 Vale: Jim, I know you were originally trained
00:18:57.07 as a physicist,
00:18:57.28 then you did beautiful
00:19:00.19 physical chemistry work,
00:19:03.07 with Eugene Kennedy,
00:19:05.18 and eventually
00:19:07.16 you managed to mature
00:19:09.07 your research problem
00:19:10.06 in vesicular trafficking
00:19:11.22 to the point where you were able to get
00:19:15.24 purified membranes and purified proteins
00:19:19.01 SNARE proteins, and reconstitute,
00:19:21.29 a membrane fusion reaction.
00:19:24.27 So that must have been a very satisfying
00:19:27.02 moment and I was wondering if you could explain
00:19:29.02 how you got to that particular
00:19:32.08 transition where you could study
00:19:35.18 this problem in this really well-defined manner.
00:19:38.08 The history of that work
00:19:40.20 was we were left logically with NSF, SNAP,
00:19:43.28 and SNAREs as components of the fusion
00:19:46.05 machinery.
00:19:47.01 Bill Wickner did an experiment
00:19:49.23 in his vacuole fusion system
00:19:51.10 that established actually that NSF
00:19:55.19 doesn't participate directly in...
00:19:58.15 NSF and SNAPs, do not directly participate
00:20:00.20 in the fusion reaction per se.
00:20:02.08 When put together with our earlier finding
00:20:05.09 that the ATP hydrolysis by NSF
00:20:07.06 disrupts the SNARE complex,
00:20:08.14 he essentially educated us,
00:20:11.21 somewhat reluctantly I might add
00:20:12.27 that NSF must be... while it is critical
00:20:18.22 to energize the system
00:20:19.20 is acting either after the fusion reaction
00:20:22.10 or prior to separating
00:20:25.06 pre-existing SNARE complexes.
00:20:26.11 In any case it left the SNAREs
00:20:27.19 pardon me, as the only
00:20:29.25 logical candidates. So with that in mind,
00:20:31.16 I would say we weren't surprised
00:20:34.10 that the SNARE complex
00:20:35.26 mediated fusion. And later,
00:20:38.01 that year Reinhard Jahn
00:20:40.05 and Axel Brunger had published
00:20:43.08 the X-ray structure
00:20:44.11 that showed a hairpin arrangement
00:20:45.26 and so it all really came together
00:20:47.13 in that respect in 1998.
00:20:50.06 And you might say that since then
00:20:51.24 we've been trying
00:20:52.12 to study more or less the physical chemistry.
00:20:54.19 My current focus is
00:20:58.00 the still unresolved problem of exactly how
00:21:01.07 calcium, synaptotagmin,
00:21:03.10 and complexin,
00:21:04.06 put a brake on fusion
00:21:05.07 at the last millisecond
00:21:06.10 in the case of the synapse.
00:21:07.13 And how they orchestrate
00:21:09.19 in a highly
00:21:10.28 amplified and synchronous fashion
00:21:12.01 the release of the neurotransmitters.
00:21:14.04 That also is a problem in physical biochemistry,
00:21:16.15 which I find enormously satisfying.
00:21:19.28 So it's sort of fun for me
00:21:21.17 towards the end of a
00:21:24.09 relatively long career,
00:21:26.01 it’s been forty years almost
00:21:27.08 in research since I left university
00:21:33.01 to come back
00:21:34.17 and have an opportunity
00:21:37.24 to really take this problem
00:21:38.27 to the structural and biochemical level
00:21:40.12 Vale: So I am curious
00:21:42.27 when you first became
00:21:44.16 aware of each other's work
00:21:46.00 and also while all these
00:21:48.13 exciting developments were happening
00:21:50.05 in each of your own labs,
00:21:51.21 were the two of you in communication
00:21:53.00 with one another?
00:21:53.26 Schekman: We were in touch with each other.
00:21:55.28 I mean after all,
00:21:56.16 Jim was at Stanford
00:21:57.11 and I was at Berkeley.
00:21:58.09 We actually had one or two
00:22:00.29 day long joint group meetings
00:22:02.15 Rothman: Two.
00:22:03.05 Schekman: Was it two?
00:22:04.06 There was some tension
00:22:06.22 between us at the time.
00:22:07.29 I think it was because
00:22:08.28 you know, we both wanted to make our name
00:22:10.28 and you know,
00:22:13.09 it was a new area,
00:22:14.12 and we were carving out
00:22:15.06 our own approach, and that was...
00:22:19.12 Rothman: But we still were very
00:22:20.07 much in communication.
00:22:20.28 It was very open.
00:22:23.10 And I'll tell you something that's a little
00:22:24.22 different today.
00:22:26.03 Oftentimes, there were never times,
00:22:28.25 when you didn't share an idea with me
00:22:30.12 that you were thinking about.
00:22:31.22 We weren't doing exactly the same experiments.
00:22:33.17 And I would always feel comfortable talking to you
00:22:36.11 about something that I was thinking about,
00:22:38.09 even before we started the experiment.
00:22:39.06 Because, you know, there's just
00:22:42.12 a certain pride of craftsmanship
00:22:44.18 and respect that really good colleagues
00:22:47.03 have even if they are
00:22:47.22 competitive with each other.
00:22:48.27 I think we always had that.
00:22:51.23 But there was still some tension.
00:22:53.22 And I think that dissolved
00:22:57.21 later on, especially,
00:22:59.13 when it became clear that we
00:23:02.04 had identified genes that
00:23:03.10 were coding proteins
00:23:05.02 that we could study.
00:23:06.05 At that point, it actually...
00:23:08.17 we both felt that we could establish ourselves
00:23:11.14 at the same time, but
00:23:14.02 through a different approach.
00:23:15.24 Vale: So obviously both of you
00:23:17.25 have had incredibly exciting careers in the field of
00:23:21.08 membrane trafficking.
00:23:23.14 If you had a chance to do it all over again,
00:23:25.23 right now, would this be the scientific
00:23:29.13 problem that you would pursue?
00:23:32.03 Schekman: Jim and I have had this conversation,
00:23:33.10 which is if we were young,
00:23:36.29 again would we start in this field
00:23:39.23 all over again? And you know, in 2013.
00:23:44.05 And the answer for very personal reasons,
00:23:46.06 is no. I would not.
00:23:48.11 And I think Jim shares this feeling.
00:23:50.14 Jim: Yeah, I wouldn't either.
00:23:51.08 Schekman: Because you know,
00:23:51.28 it's a relatively mature field.
00:23:54.05 I think there are many interesting things to discover.
00:23:55.19 But, you know,
00:23:58.19 you heard what I said earlier.
00:24:00.10 I wanted to make my own identity.
00:24:01.25 I didn't want to be
00:24:02.18 in someone else's shadow.
00:24:04.04 And so for that reason,
00:24:05.23 if I were starting over again
00:24:07.19 I would pick something brand new
00:24:10.02 that was underexplored, and where
00:24:13.17 I'd have a lot of time
00:24:15.05 and freedom to develop
00:24:18.07 a novel approach and make new discoveries,
00:24:22.12 really significant new discoveries.
00:24:24.24 So I don't mean to discourage
00:24:26.20 people from thinking that way,
00:24:28.14 Jim: If I could say at another level,
00:24:30.09 I totally agree with what you said, Randy.
00:24:32.07 But it all depends on
00:24:33.23 your scientific focus.
00:24:34.24 So if I was a young...
00:24:36.05 I am a professor of chemistry at Yale.
00:24:38.17 I am getting a lot of interest
00:24:39.21 in my work now from the chemists.
00:24:41.19 You know, because at another level,
00:24:45.00 this is now a problem
00:24:46.10 that where the biology is considered
00:24:48.17 relatively solved.
00:24:49.12 Nothing is ever completely solved.
00:24:51.07 Nobel Prizes aren't given out for stuff
00:24:54.09 that is basically solved.
00:24:55.06 On the other hand, it's a problem that has just been defined
00:24:57.29 from the point of view
00:24:59.04 of the physical biochemistry
00:25:00.20 and the chemical biology
00:25:01.25 that can come from it.
00:25:02.24 So depending on your world view,
00:25:05.06 for cell biologists or developmental biologists,
00:25:07.09 there's probably other things to do
00:25:09.04 that are really bigger challenges today.
00:25:10.23 For chemists, physical chemists, even physicists,
00:25:15.11 and people interested in modeling
00:25:17.17 it's really, really primary.
00:25:20.18

This Talk

Speakers: James Rothman, Randy Schekman

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: December 2013

Talk Overview

Randy Schekman and James Rothman were awarded the 2013 Nobel Prize in Physiology or Medicine for their work on elucidating the components and mechanisms of the secretory pathway. In an interview with Ron Vale, Drs. Schekman and Rothman talk about their early careers and the key experiments that led to some of their most significant discoveries.

Speaker Bio

James Rothman

Dr. James Rothman is the Wallace Professor of Biomedical Sciences and Chair of the Department of Cell Biology at Yale University School of Medicine. He uses biochemical and biophysical approaches to uncover the mechanisms involved in vesicle trafficking and vesicle fusion. In addition to the Nobel Prize, Rothman has received many awards and honors for… Continue Reading