AUSTRALIAN FRONTIERS OF SCIENCE, 2005
Walter and Eliza Hall Institute of Medical Research, Melbourne, 12-13 April
Machinery for mitochondrial protein import on the roads to ruin
Associate Professor Trevor Lithgow, Department of Biochemistry and Molecular Biology, University of Melbourne
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I want to split my talk today into two parts. The first part is to describe for you the fundamental work that we do on protein targeting, which is our main game in the lab. Then in the second part of the talk I want to relate to you a couple of collaborative studies that we have undertaken coincidentally, one with David Vaux and one with David Huang where we have used some of the knowledge that we have gained on how proteins are targeted to mitochondria, to try and really get to grips with the mechanistic details of how some of the regulators of programmed cell death, a really important applied problem as David Huang has described to you, can be regulated.
Firstly, there are two essential subcellular compartments that are found in all eukaryotic cells. One is the nucleus and the other is the mitochondria. And it is the mitochondria that we are especially interested in understanding. That is shown on the left of this slide in a transmission EM micrograph taken out of an undergraduate textbook. The cartoon on the right perhaps explains it a bit better. It represents the submitochondrial structure by which around 1000 different proteins from these eukaryotic cells are compartmentalised into the matrix – the central, soluble space of the compartment; the inner and the outer membrane; and, in addition, the soluble space between those two membranes. Of these 1000 or so different proteins, 99 per cent are actually made outside the mitochondria. They have to be imported into the mitochondria and then sorted appropriately, so that each protein has a single submitochondrial destination.
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If we zoom in on that picture and take it cartoon-style, you see here the outer membrane of the mitochondria, the inner membrane, the intermembrane space and the matrix compartment. What I want to talk to you about in the first part of this talk is how we now understand protein import into mitochondria to work. What we have come to realise is that there is a series of molecular ‘machines’ which are responsible for recognising all the proteins made outside the mitochondria, allowing them either to access or not to access the translocation machinery, depending on whether they are or are not mitochondrial proteins, to translocate those protein molecules across the outer membrane and then to do a job on them to sort them, either into the intermembrane space or into the outer membrane, the inner membrane or the matrix. So this is a series of molecular machines which are responsible for this crucial biological process.
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I want to focus my attention today on the TOM (through the outer mitochondrial membrane) complex, partly to limit things for the sake of time and partly because this complex is the molecular machine that we have spent most of our research effort on coming to understand. The TOM complex represents the only portal into the mitochondria, so all protein import has to go up by way of this complex, which is built up from a series of integral membrane proteins.
There are four fundamental components that we understand the function of, and I will zip through those in the next few slides. There are also a couple that we are still trying to understand. As I said, it represents the only way for proteins to gain access to the mitochondria, so all of the thousand proteins that go to make up the mitochondria have to enter the mitochondria through this TOM complex.
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I will give you a couple of slides on the components of this molecular machine that we understand in the most detail. The first is Tom20 – and again for the sake of time I will limit the explanation and the data to things that we have done ourselves, though I would be quick to add that there are a number of groups around the world that are working on a similar problem. In our lab we have used NMR, we have used bioinformatics and we use a range of cell biology assays, and we have applied them to trying to understand the function of Tom20. We now feel confident to say that Tom20 is the component of this molecular machine that does the recognition: it recognises every one of the thousand different proteins that have to get into the mitochondria.
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We know from structural analysis that there is a receptor domain on Tom20 – in fact, it forms most of the body of the protein, which is composed of a series of helical segments that are stacked together in such a way that they create a cradle into which the targeting sequence that designates a protein as mitochondrial can be bound. That receptor domain is then linked to the outer membrane by virtue of a flexible disordered region of polypeptide that allows the receptor (we would imagine) to manoeuvre around the substrates that it might bind, and then a transmembrane segment. So it is an integral membrane protein and part of this component of the molecular machine sits within the phospholipid bilayer of the mitochondrial outer membrane.
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We have looked at a second component of this molecular machine, Tom70, here using small-angle X-ray scattering instead of NMR so far we have had more joy with that technique but again bioinformatics and cell biology, to show that Tom70 is responsible for binding the very hydrophobic parts of most of the proteins that get to the mitochondria.
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In this sense it cooperates with Tom20 in the recognition process. We know that Tom70, if you look at an arrangement of the protein’s outline, domain structure, just stretched out from amino to the carboxy terminus, is made up of a series of repeat units – the little grey boxes here represent structural repeats called TPRs, and there are, we would imagine, 11 in all versions of Tom70 – which zigzag backwards and forwards on top of each other to create a long meandering molecule that the SAXS analysis would say is actually folded back into three distinct lobes or domains of Tom70. We are currently trying to work out exactly how it is that this structure is capable of binding all of the very hydrophobic parts of all these various mitochondrial proteins.
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The last two components that I want to introduce to you today are Tom22 and Tom40. Here we know that Tom40 is a beta barrel structure that actually structurally forms the translocation channel through the mitochondrial outer membrane. It is through the central cavity of Tom40 that all proteins have to travel if they are to cross the outer membrane, and Tom22 is bound tightly to this channel.
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In fact, we have found Tom22 sequences from essentially all of the eukaryotes you would like to look at – from plants, from animals, from fungi, and from all sorts of human pathogens representing the more weird eukaryotic organisms that you can find.
I won’t go through it in detail, but the sequence analysis here simply says that the region within the box, which is the region of Tom22 that sits within the membrane, has a number of key residues that are very tall on this plot. What that means is that they are absolutely conserved across evolution. The P peak here stands for a proline residue within Tom22, and the fact that it is tall enough to fill out the box says that there is a proline residue found at that position in every Tom22 from every eukaryotic organisation that we have sequenced and can analyse. And we know from mutagenesis studies, which again I won’t go into in detail, that this proline is part of the contact face that Tom22 makes with Tom40.
So we imagine that Tom22 actually sits up against Tom40 in this way, and thereby exposes part of itself to the cytosol, where it can help Tom20 and Tom70 in recognition of mitochondrial proteins, and displays part of itself within the intermembrane space where this part in the intermembrane space, we believe, acts as a kind of entropic spring, a relatively novel mechanism for mediating the transfer of the protein through the guts of Tom40 and making it available to proteins within the intermembrane space that will then continue the journey of this substrate through the mitochondrial compartments.
So what I want to convince you of, albeit very briefly, is that we have assays in place where we really can understand the components of the protein import pathway into mitochondria. I think we have been relatively useful so far in helping people like David Vaux and David Huang in their understanding of how proteins that affect major biological pathways can in fact be activated by mitochondria. So I want to give you two examples by which our understanding of the way that proteins are targeted and processed within mitochondria have helped us in understanding these important biological processes.
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I will firstly talk about a protein called DIABLO, and then secondly I will come back, albeit very briefly, to mention again some of the proteins that David Huang has already talked about, the Bcl-2 family that his lab works on, and the way in which we think the mitochondria are responsible for assisting their activation during the course of programmed cell death.
The first one, DIABLO, is a protein that was first identified by Anne Verhagen and David Vaux and their collaborators, including Richard Simpson at the Ludwig Institute, a few years ago now. It was clear from the very first identification that DIABLO is a protein that can impact on cell death – in fact, precipitate cell death – and it does so by interaction with a set of inhibitors called the BIR type of proteins. I won’t go into the details; it is certainly not my area and we don’t have the time to go through it. But DIABLO was already known, from its first identification, to be interacting with this group of proteins called the BIR-type inhibitors, and the interaction was mediated by the amino terminal residues of DIABLO.
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Let’s look at one of these BIR-type domains, as shown here in grey, this blob representing the surfaces of one of these proteins. From structural work by a couple of groups overseas it is clear that there are a couple of pockets – a shallow pocket and a deeper one – into which the amino terminus of DIABLO inserts its amino acid side chains. The side chain from the A here, representing an alanine, is sitting in the shallow pocket, and the large side chain from the I, representing an isoleucine, is sitting into the deeper pocket. In fact, the sequence AVPI is absolutely crucial to allow DIABLO to mount both of those pockets and make the appropriate contacts so that it can precipitate cell death.
What you will also notice, if you are keen, is that the alanine at the terminus of DIABLO is actually no. 54 in the polypeptides. DIABLO is made as a precursor with an additional 53 amino acids that are not needed for its function and in fact will block its function if they remain intact. So we were interested to understand the nature of the protease that is responsible for cleaving away those 53 inhibitory amino acids, thereby activating DIABLO.
So how is this done?
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Well, again from previous work, including the work that Ann and David did, we knew DIABLO was found in the mitochondria that was why we got involved in the project in the first place and so we knew that the enzyme responsible for the processing would be a mitochondrial protein. We looked in yeast, where we knew the complete genome sequence, we knew all of the proteases that can do protein processing and we knew where they all lived, and there are 32 mitochondrial proteases so it is a relatively short list of things to check through. Working in yeast it is relatively straightforward to make mutants and so we could check a number of candidates, and we were ready to sift through all 32. But early on in the search we realised that two proteases, Δimp1 and Δimp2, were responsible for processing DIABLO. They could take the long form, clip off the 53 amino acids and convert it to the short form. And if you knock out either Δimp1 or Δimp2 then you stop the yeast from being able to activate DIABLO.
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Putting this back in the context of mitochondrial protein import and together with a lot of data that I won’t show: we now know that DIABLO goes through the TOM complex – it interacts with Tom20 and Tom70, and goes through Tom40 – and ends up on its way to the inner membrane. But along the way the IMP complex, made up of Δimp1 and Δimp2, which we know is in association with one of these other molecular machines, cleaves DIABLO, takes away the 53 inhibitory amino acids and leaves it resident in the mitochondrial intermembrane space, ready to act on programmed cell death.
Finally I want to talk about the project that we have just initiated with David Huang, looking at the activation of the Bcl-2 family of proteins.
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At least some of the members of the Bcl-2 family of proteins are targeted to the mitochondria and in fact they are able to be inserted into the mitochondrial outer membrane. Most outer membrane proteins are made, as I said, in the cytosol; they have to be imported by the mitochondria. And mostly they would make use of the TOM complex and the next molecular machine, the SAM (sorting and assembly machinery) complex, to be inserted into the bilayer.
What is curious, and in fact biologically really important about the Bcl-2 family of proteins, is that they don’t make this entire journey until the cell has received signals for cell death. And so they are stalled, either prior to their interactions with the TOM complex or perhaps even after their interactions with the TOM complex but before they have been fully assembled, in the mitochondrial outer membrane. We want to apply some of our knowledge of these molecular machines to try and understand how it is that the activity of the Bcl-2 family of proteins can be stalled in this way.
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So, firstly – again I apologise but I won’t go through the details of this – this is simply a native gel electrophoresis assay to prove to you that we do have some sensitive assays for studying the process. And most outer membrane proteins start off as a precursor form and end up as a mature form. In fact, in wild-type mitochondria you can follow very rapidly the precursor being converted to the mature form of the protein after assembly into the outer membrane. Indeed, if we go through and have a look at some of our yeast mutants – we do have a collection of them – we see that some of them are decrepit in their ability to insert these proteins into the outer membrane. Some of the mutants affecting the TOM complex can stall the process; some of the mutants affecting the SAM can stall the process. And we want to use the assays and also our collections of mutants to study David’s proteins and try and understand how it is that these proteins are actually biologically stalled along the way to their insertion into the outer membrane.
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Looking at the structures of what probably represents the precursor form of these Bcl-2 family of proteins: this figure is actually taken from a paper by Mark Hinds, Catherine Day, David Huang and their collaborators, and is simply to show that, in its precursor form, some of these Bcl-2 type proteins like Bcl-w, Bax and Bcl-XL, sit in a soluble state – so these are NMR structures, the protein was soluble when it was analysed – and their C-terminus (a rather hydrophobic helix of the polypeptide which here has been coloured in blue) is folded back on this soluble protein so that the entire three-dimensional structure is quite happy to exist in a soluble monomeric form. We would imagine that this blue, very hydrophobic part of the protein, lying back on the rest of the protein, would represent the precursor form of these Bcl-2 type proteins before they get into the mitochondrial outer membrane.
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What we are now setting out to do is to try and understand whether this precursor form, with its blue hydrophobic part lying back against the protein, is simply flicked open, into a more open conformation, and – sort of switch-blade style – this blue hydrophobic part can then be plunged into the outer membrane, perhaps using the TOM complex, perhaps also using the SAM complex. And then the key trigger for precipitating cell death would be the molecules which are able to pull this hydrophobic part out of the protein and plunge it into the outer membrane.
Another potential model, perhaps less attractive but only because it is much more complicated, is that the soluble form of these factors has to be completely reorganised and that the blue part, along with all the rest of the polypeptide, is stitched into the outer membrane in multiple transmembrane segments. Certainly the mitochondria is capable of doing this. Many of the outer membrane proteins do have this sort of topology, and the TOM and the SAM complexes can force a polypeptide to do this. And we do think that the assays that we have will enable us to distinguish between the two models and to really come to some mechanistic understanding of how it is that this family of proteins gets targeted to mitochondria and assembled there in their active forms, and can thereby impact on programmed cell death.
So I just want to reiterate, very quickly, the three main points that I made. Firstly, the process of protein import into mitochondria is mediated by a series of molecular machines made up of independent component parts, most of which we now understand the functions for. Secondly, we have assays which have enabled us to understand the functions, the mechanistic detail, of these machines, which are now really useful in terms of teasing apart other biological processes that the mitochondria impact on. And, thirdly, one of the examples, the one that we have been most involved in studying with David Vaux’s lab and also David Huang’s lab, is the process of programmed cell death.
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Lastly, I would like to say some thanks. I didn’t go through and talk about all the work that all the people here have done, but the work on Tom70 is Nickie Chan’s PhD work; the work on Tom40 is Michael Dagger’s PhD work; and the work on Tom20 has been a collaboration between Joanne Hulett and Andrew Perry. (Andrew was away for the photograph.) I also want to thank Paul Gooley, Vladimir Likic and Terry Mulhern, who are all members of the Biochemistry Department at Melbourne University, at the Bio21 Institute of Molecular Science and Biotechnology. And the three collaborators shown on this slide are our structural advisers, who in many cases also do the actual structural analysis on our TOM proteins.
For cell death we have a number of collaborators: Chris Hawkins, up at the Murdoch Institute (I didn’t have a chance to talk about the projects we have worked together on) and also David Vaux, David Huang, Jamie Fletcher and Ruth Kluck, here at the Walter and Eliza Hall Institute.
I would also like to thank the ARC,
who support all of our work; the Human Frontiers Science Program, who have
supported some of the previous work; and also the organisers for this session,
for inviting
me along.
