Discovery of Chaperonin-Assisted Protein Folding

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00:00:07;07 Hi, I'm Art Horwich, from Yale School of Medicine and HHMI.
00:00:10;13 And I want to tell you a story about a discovery that has led to a really wonderful
00:00:17;29 what's now a fifteen to twenty year experience of experimentation that followed.
00:00:23;19 This discovery was really somewhat serendipitous,
00:00:27;09 and I think there was a lot of luck involved.
00:00:29;09 It was helped enormously by two wonderful collaborators, who have remained very close friends,
00:00:36;09 and constant contacts, Ming Cheng, who's now in Taiwan,
00:00:41;12 and Ulrich Hartl, who's now in Martinsried.
00:00:44;13 And so I'll just tell you the story as it occurred.
00:00:48;09 So, the story is about information transfer in the cell.
00:00:52;16 As we know, DNA is transcribed into RNA and is in turn translated into protein
00:00:57;12 and the step that's involved here is the final step of information transfer,
00:01:03;08 namely that of protein folding.
00:01:06;01 So, we know about machines that carry out transcription and translation,
00:01:10;05 and have very fine molecular details to those reactions for some years now,
00:01:16;22 increasingly fine.
00:01:19;11 But protein folding has remained somewhat mysterious
00:01:23;14 and it's really only during the last decade or two
00:01:25;18 that we've really started to gain some understanding about how this works in cells.
00:01:31;23 So, for those unfamiliar, I'd just point out that proteins are composed of twenty different amino acids,
00:01:38;17 and these strings of amino acids ultimately fold into characteristic three-dimensional structures
00:01:44;18 that carry out biological activities.
00:01:47;03 For example, they can be enzymes, cytoskeleton, hormones, hemoglobin, channels,
00:01:52;18 receptors, what have you.
00:01:54;15 Proteins are major effectors in the cell
00:01:57;21 and so their proper folding is crucial to them being biologically active,
00:02:03;08 because when they're effectively strands of spaghetti,
00:02:07;00 leaving a ribosome, they don't necessarily have any biological activity at that point.
00:02:13;08 They have to be folded correctly in order to be active.
00:02:16;24 And so the granddaddy experiment of protein folding
00:02:20;00 that I heard about when I was an undergraduate at Brown University
00:02:24;04 in 1972 was carried out by Christian Anfinsen, in fact, in the late 50's,
00:02:30;29 he received his Nobel Prize for this work in 1972,
00:02:35;27 a really astonishing experiment where he started with an enzyme,
00:02:39;23 ribonuclease, 100-odd amino acids with 4 disulfide bonds,
00:02:44;05 and unfolded that protein by treating it with denaturant, urea,
00:02:48;20 and reductant, mercaptoethanol.
00:02:50;28 That produced essentially a random coil version of the protein,
00:02:54;26 and he asked whether, when he removed the urea and reductant,
00:03:00;14 whether the protein could find its way back, on its on, in a test tube,
00:03:04;21 to the native active form, and astonishingly, it did so.
00:03:09;01 So, this proved that a polypeptide chain contains all of the information
00:03:14;12 for folding into the unique native structure in its primary amino acid sequence structure.
00:03:20;21 He also presumed that in fact this native state would lie at an energetic minimum.
00:03:27;17 And so, this astonishing experiment led people to believe that perhaps inside cells,
00:03:36;19 proteins also fold spontaneously.
00:03:39;13 But increasingly, there were instances where that didn't really happen,
00:03:44;03 both in test tube, where many proteins could also not be spontaneously refolded,
00:03:50;05 as in the Anfinsen experiment, with ribonuclease,
00:03:53;18 but inside cells when the biotech industry began to express mammalian proteins in E. coli,
00:03:58;28 for example, they also saw these terrible looking aggregates with no biological activity
00:04:04;27 and failed to recover their favorite proteins in a native, active form.
00:04:09;13 And this, once again, one saw, as in the test tube,
00:04:13;26 a misfolded, aggregated, inclusion body, as it were.
00:04:18;21 And so this suggested that something more might be required in the cell
00:04:23;24 to deal with these so-called, what would be kinetic problems of folding,
00:04:30;10 where proteins simply get it wrong and go off pathway
00:04:34;07 and misfold and aggregate.
00:04:36;00 And so we never thought we would have anything to do with this,
00:04:40;18 but we were busy in the late 80's studying mitochondrial protein import.
00:04:48;19 So, it turns out that most of the proteins of mitochondria are encoded in the nuclear genome,
00:04:54;22 the messages for these proteins are then translated on cytosolic polyribosomes
00:05:02;17 and an N-terminal targeting peptide in these proteins directs them to mitochondria.
00:05:10;06 And they're then imported through the mitochondrial membranes,
00:05:14;16 and the targeting peptide is then clipped and the mature-sized protein
00:05:19;00 then has to fold into its biologically active form.
00:05:22;29 Well, an important experiment was carried out in 1986 by Gottfried Schatz and his coworkers
00:05:28;18 that showed that a protein had to be unfolded in order to transit the mitochondrial membranes.
00:05:35;19 He used dihydrofolate reductase as a substrate in these experiments,
00:05:41;03 showing that if you lock DHFR into its native state,
00:05:44;22 by adding the ligand methotrexate, the protein couldn't go through the membranes,
00:05:49;19 whereas, if you now removed methotrexate or denatured the protein,
00:05:54;06 it would go through the membranes.
00:05:55;25 So, the question that arose to us, as we were studying mitochondrial protein import,
00:06:01;05 was, do proteins, upon reaching the mitochondrial matrix space,
00:06:07;03 refold spontaneously to their active form, as might be suggested by the Anfinsen experiments,
00:06:13;12 or could there be a machinery that was required to assist the protein to reach its native, active form.
00:06:20;27 And so we were in a position to actually test this
00:06:24;11 because we had a bank of temperature sensitive lethal yeast mutants
00:06:28;28 that contained a galactose regulated version of a mitochondrial protein,
00:06:34;20 ornithine transcarbamalase, a human protein that we had shown in yeast already
00:06:39;10 could be expressed and would go faithfully to its mitochondria, have its signal peptide cleaved,
00:06:44;12 and become biologically active and make OTC enzymatic activity.
00:06:48;05 And so our screen was basically to start with temperature sensitive lethal yeast mutants,
00:06:54;11 the idea being from Schatz and others in the field
00:06:57;03 that if you can't make new mitochondrial proteins and new mitochondria,
00:07:02;10 the cell basically stops its growth and ultimately, it's a lethal.
00:07:07;09 So, we start with a bank of lethal mutants,
00:07:10;12 programmed to express mitochondrial OTC behind a gal operon promoter,
00:07:16;03 and we proceeded to test each mutant individually for the ability to make OTC activity
00:07:21;28 after shift to non-permissive temperature and shift into galactose,
00:07:26;05 to turn on OTC expression under non-permissive conditions.
00:07:30;13 We then analyzed OTC activity; if it wasn't present, we would then Western blot
00:07:36;20 the particular mutant after shift to 37 C and induction of OTC to see what the fate of the OTC subunit was.
00:07:44;09 Was it present as a mature size protein, or was it still a precursor
00:07:48;11 that hadn't been recognized by mitochondria and so our idea was to dissect the mitochondrial import pathway.
00:07:54;26 Well, as we were asking this question about refolding inside the mitochondrial matrix,
00:08:01;28 we knew what we would be looking for in the form of a mutant
00:08:05;05 that affects refolding in the mitochondrial matrix space.
00:08:08;20 We would have mature size OTC subunits because their signal peptide would be cleaved
00:08:13;29 and they'd be inside the matrix space, but we would have no enzymatic activity,
00:08:18;26 because they would have failed to correctly fold, and in the case of OTC,
00:08:22;23 trimerize into the active form of the enzyme.
00:08:26;20 So, we started to look through our bank of mutants
00:08:31;12 and to our astonishment, almost right away, having asked this question,
00:08:35;23 we found a mutant in which exactly that happened.
00:08:38;20 So, OTC was our initial reporter enzyme and what we found was that OTC subunits entered,
00:08:45;20 they reached their mature size,
00:08:48;03 and then there was no biological activity at all.
00:08:50;28 So, this astonished us.
00:08:53;20 It seemed like heresy to have a result like this.
00:08:56;18 So, we though, uh huh, we better start to look at endogenous yeast proteins
00:09:01;27 and see what is happening to them.
00:09:04;04 So, we looked at this particular protein, the F1 beta subunit of ATPase,
00:09:09;05 of the F1 ATPase.
00:09:11;02 And we found that in fact, similarly, this beta subunit had failed to assemble
00:09:17;26 into this stalk like structure and the subunits themselves looked like had misfolded
00:09:24;06 in the mitochondrial matrix space.
00:09:27;11 We were in a bit of a quandary, because, again, it seemed like heresy,
00:09:33;06 and we couldn't really feel comfortable about the data at hand
00:09:38;04 and we were sort of pondering what proteins to look at next
00:09:42;12 when thankfully, the phone rang and it was Ulrich Hartl and Walter Neupert
00:09:47;03 calling from Munich to say, well, we understand that you guys have some mutants of yeast
00:09:52;23 that affect mitochondrial protein import
00:09:55;11 and we would love to get together with you and perhaps help on some of the biochemistry of those mutants,
00:10:01;12 if you'd have some interest.
00:10:03;08 So, at that point in history, my lab had four people in it,
00:10:07;18 and we weren't very well versed at fractionating yeast mitochondria,
00:10:12;29 whereas these were world experts,
00:10:15;09 so we were just delighted to interact and I went to Munich the next week
00:10:20;29 and presented some of the better described mutants that we had
00:10:25;12 that affected the mitochondrial signal peptide processing.
00:10:29;08 But I also, at the end of my talk, described this particular mutant,
00:10:33;13 and Walter and Ulrich were somewhat worried that what was happening in this mutant
00:10:39;07 was that proteins were coming in through this contact site
00:10:43;08 but weren't getting entirely into the mitochondrial matrix,
00:10:47;01 that they were poking their N-terminal signal peptides in and the signals were being cleaved,
00:10:52;04 but then the proteins weren't entirely entering
00:10:54;18 and of course, they wouldn't fold if they couldn't enter through the membranes.
00:10:58;12 And so, I said, well I'd be absolutely delighted to send you the mutant
00:11:02;29 and you can make mitochondria, I am sure, from this mutant, very effectively,
00:11:08;06 and analyze to see what's going on and in fact, as soon as I got back, I sent the mutant to Munich,
00:11:16;10 and two weeks later, Ulrich called up to say, excitedly, you're right,
00:11:22;05 the proteins are entirely in the mitochondrial matrix, I can add exogenous protease,
00:11:27;17 and it doesn't touch them, so it seems like they have gone into the matrix space
00:11:32;10 and they have failed to fold correctly.
00:11:35;06 So, this was a really wonderful time where there was a lot of back and forth,
00:11:39;16 with Ulrich and I crossing the Atlantic to visit each other and discuss the phenotype of this mutant.
00:11:47;07 This is us together at his parents' house,
00:11:51;03 here's his lovely wife Manajit, who is a biochemist herself
00:11:55;13 and has worked some on the GroEL system as well, in recent times,
00:11:59;13 a chaperonin of bacteria.
00:12:02;03 And maybe this is her telling us how this is all going to work out.
00:12:06;00 But in any case, we carried out together a set of further experiments
00:12:13;15 on a number of mitochondrial matrix proteins
00:12:16;12 to see what was happening to them.
00:12:18;18 And one of the most exciting results came with this particular protein,
00:12:22;12 the Rieske iron sulfur protein.
00:12:24;29 So, this is a protein that is imported to the matrix space and has to be cleaved twice
00:12:30;24 as a monomer, before it enters the inner mitochondrial membrane.
00:12:35;18 And when we looked at this iron sulfur protein,
00:12:38;19 we found it in either a completely uncleaved state
00:12:42;24 or in a once-cleaved state, as if the protein had not folded correctly
00:12:47;00 and had not undergone the proper cleavage that enables its correct biogenesis.
00:12:52;01 So, this was the first of a monomeric protein where we saw effects in this particular mutant.
00:12:58;02 We, at this point, we rescued the mutant with a yeast library.
00:13:04;12 So, we put on a bank of yeast clones and the gene that rescued was sequenced
00:13:12;07 by Ming Cheng during, I believe it was summer 1988.
00:13:17;06 And at this point, we knew that there was a modestly heat inducible protein
00:13:23;02 inside mitochondria that had been identified the year before by Richard Hallberg,
00:13:27;26 working in Iowa, in Tetrahymena.
00:13:30;11 And we had a feeling that Hallberg was analyzing the sequence of the corresponding homologue in yeast
00:13:38;06 with a view to doing further experiments in that system.
00:13:41;16 So, on a Saturday afternoon in that summer, Ming Cheng called up Richard Hallberg
00:13:48;12 with, to see whether her sequence of this gene would match the sequence that he had
00:13:54;18 of the homologue he was studying.
00:13:56;22 Well, I had gone to JFK airport to pick up Ulrich,
00:13:59;28 because the two of us were going to a Gordon conference on mitochondria the next day,
00:14:03;28 and my son came with us, it was many hours, but we got back to the, to our little beach house
00:14:10;18 at around 6 o'clock in the evening and there's Ming Cheng and the rest of the lab,
00:14:15;01 standing in our front yard and Ming's just jumping up and down holding a sequencing gel,
00:14:20;00 saying, "it's a dead match, it's a dead match!" and we said, well, what's the dead match?
00:14:25;07 She said, oh, I called up Hallberg, our protein corresponds to this ring assembly that he's studying!
00:14:31;10 And so, two and two came together in a really amazing moment and we had a wonderful evening of celebration,
00:14:38;00 but basically, what it amounted to was what we then called heat shock protein 60,
00:14:44;05 collectively, with Richard and Ulrich and Walter and everybody.
00:14:48;26 This modestly heat inducible protein
00:14:52;29 was actually assisting the folding of all these proteins being imported in the mitochondrial matrix.
00:14:59;16 And in fact, however, Hsp60 is essential at all temperatures,
00:15:06;08 so its assistance to polypeptide chain folding is required under normal conditions,
00:15:12;01 which embody, often, relatively high temperatures,
00:15:15;25 and very high solute concentrations in the mitochondrial matrix,
00:15:19;13 ergo this machine is essential under all conditions.
00:15:22;27 Now, shortly after this, Ulrich carried out a beautiful experiment with Joachim Ostermann
00:15:28;03 where they analyzed another monomeric protein, dihydrofolate reductase.
00:15:33;14 And what they could see was that when they put a signal peptide on DHFR to make it go into mitochondria,
00:15:39;04 much as in the Schatz experiments,
00:15:41;13 letting it go into mitochondria, it became associated with this complex,
00:15:46;24 with this ring assembly that Hallberg had first identified, the Hsp60 complex,
00:15:52;12 as long as ATP was relatively deprived from mitochondria.
00:15:56;15 If they added ATP back to this system, they now saw DHFR depart from this complex,
00:16:02;13 and appear as a native folded protein.
00:16:05;02 While the protein, I should say, was associated with Hsp60,
00:16:09;12 it seemed to be non-native, very protease susceptible,
00:16:12;21 recognizable by antibodies that specifically saw non-native forms of DHFR,
00:16:17;25 and so these experiments, with Rieske iron sulfur protein, with DHFR,
00:16:23;01 convinced us that Hsp60 was some sort of a folding machine in the mitochondrial matrix.
00:16:28;21 And of course, there's some generality to this because when we saw the sequence of Hsp60,
00:16:36;13 we knew already that it was going to be homologous, and is homologous,
00:16:41;14 to the GroEL protein in the bacterial cytoplasm, essential in that context,
00:16:47;05 and had been shown to be involved, or had been implicated
00:16:50;23 early as being involved in lambda phage head assembly,
00:16:53;29 now this idea had to move to lambda phage protein folding.
00:16:58;15 There was also a protein inside of chloroplasts called the rubisco binding protein,
00:17:04;04 identified by John Ellis as being required for assembly of rubisco,
00:17:08;17 which is a CO2 fixing enzyme, in fact, the most abundant protein in the biosphere.
00:17:14;01 This now, had to go to the level of thinking that rubisco binding protein
00:17:19;07 was actually mediating protein folding of rubisco subunits.
00:17:23;29 And finally, shortly thereafter, working together, Ulrich and I, with Jonathan Trent,
00:17:29;18 identified a heat shot protein in thermophilic archaebacteria
00:17:34;02 that we called thermophilic factor 55, another double ring assembly,
00:17:38;24 that is highly induced when you take these normally heat loving bacteria
00:17:43;12 and take them to an even higher temperature,
00:17:45;21 that turns out to look just like a chaperonin, and it also could turn over ATP
00:17:51;21 and behave just like these other ring assemblies.
00:17:54;24 And, in fact, when we analyze this subunit, we found a homology
00:17:59;08 to a protein in the eukaryotic cytosol called TCP1, or CCT for short,
00:18:05;00 this being a member of the eukaryotic cytosolic chaperonin 1 subunit of the 8 subunit rings
00:18:12;07 that comprise that chaperonin, essential for folding actin, tubulin, beta propeller proteins,
00:18:18;02 and many other proteins.
00:18:19;25 So, the generality of these ring assemblies in mediating protein folding to the native state
00:18:25;00 seemed to be getting established.
00:18:27;17 And so finally, mechanistically,
00:18:30;08 20 years later, we understand at least how the bacterial chaperonin system works
00:18:35;23 in pretty good detail now, with much biochemistry, X-ray crystallography,
00:18:40;28 NMR studies and a lot of EM conducted on this particular machinery, the GroEL machine
00:18:47;19 and its cooperating component, GroES, and the bottom line in terms of mechanism is
00:18:53;25 that an open ring of GroEL with all this hydrophobic surface specifically binds non-native proteins
00:19:00;03 through their exposure of hydrophobic surface that will be buried to the interior
00:19:04;22 in the native state, thus GroEL specifically recognizes non-native proteins
00:19:10;01 and once it's bound to protein, when GroES now encapsulates the same ring
00:19:15;23 that's bound by polypeptide, the polypeptide is released into this hydrophilic folding chamber,
00:19:22;18 all this blue here, signifies electrostatic residues,
00:19:26;21 and the polypeptide now folds in isolation, in a sequestered environment,
00:19:31;17 where it simply can't aggregate with any other protein and it basically uses Anfinson's principles,
00:19:38;14 its primary structure, to find its way to the native state without the fear of aggregation or going off-pathway
00:19:46;19 and being lost. So, in general, molecular chaperones,
00:19:51;23 if one wants to talk about all of the classes of these components,
00:19:55;27 they basically recognize exposed hydrophobic surfaces
00:20:01;18 that are proffered when a protein misfolds, or during biogenesis,
00:20:06;12 and the idea of the chaperone is to bind these surfaces and prevent them
00:20:12;03 from self-associating with each other in multi-molecular aggregates.
00:20:16;13 The idea of all of the chaperones is to prevent aggregation
00:20:20;19 by binding these surfaces in various geometric contexts.
00:20:24;05 The remarkable thing about the chaperonins, however,
00:20:27;00 that's really unique, is that they use nucleotide to release the polypeptide
00:20:32;13 into a sequestered folding chamber where the polypeptide really has no chance of aggregation
00:20:38;08 and has a chance to fold using its own primary structure
00:20:42;11 to the native state, one of nature's really most beautiful machines,
00:20:46;22 used for this final step of information transfer.
00:20:49;23 So, thanks very much for listening to this lecture, I hope you enjoyed it.
00:20:54;05 Thank you.

This Talk

Speaker: Arthur Horwich

Audience:

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Recorded: July 2012

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Talk Overview

Dr. Horwich explains that his lab was studying the import and folding of mitochondrial proteins in yeast mutants when he, and his collaborator Ulrich Hartl, discovered Hsp 60, a mitochondrial matrix protein required for proper folding of imported proteins. Hsp60 was found to have homology with proteins from bacteria and plants leading to the discovery of the molecular chaperonin protein family and its role in protein folding.

Speaker Bio

Arthur Horwich

Arthur Horwich received his AB and MD from Brown University. Following a pediatric residency at Yale, Horwich did two postdoctoral fellowships, first with Tony Hunter and Walter Eckhart at the Salk Institute and second with Leon Rosenberg at Yale, before joining the Yale faculty. Currently, Horwich is Sterling Professor of Genetics and Pediatrics at Yale… Continue Reading