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0:08
Hi. My name is Jared Rutter, and I'm a Professor of Biochemistry and an Investigator of the Howard Hughes Medical Institute at the University of Utah. And I'm gonna tell you today, in the next 30 minutes or so, some of the things that I find most fascinating about the mitochondria. So, to give you a little bit of my background and how we became interested in this organelle, I did my PhD in the lab of Dr. Steve McKnight at the University of Texas Southwestern Medical Center.
0:38
And at the time I joined Steve's lab, he had just recently come back to academia from a biotech company. And as a result of that, the lab was relatively small, and there weren't really ongoing projects. Everything was brand new. And Steve really encouraged us at that time to really focus on doing something brand new, focus on doing something unique, make a discovery that no one else would ever make. That's how we were taught to think about science,
1:07
and that's a teaching that has really influenced my career and something that I've always aspired to. This is really difficult. We all like to stay in our comfort zones, and this is a really difficult standard to hold ourselves to, but I think it's very important for us as scientists. So, after being in Steve's lab, I eventually went to the University of Utah and joined the Department of Biochemistry, and was surrounded by a bunch of wonderful colleagues,
1:35
some of which worked on this organelle, the mitochondria. Specifically, Janet Shaw and Dennis Winge really inspired me with the work that they had done to understand mitochondria and make important discoveries. And I began to be kind of intrigued and thought that my lab should maybe do something to try and understand this organelle better. And I'll tell you, one of the things that really captured my attention -- relating to how Steve taught us to think about science --
2:02
is the degree to which there were many things about mitochondria that we just didn't understand well... about this complex structure in cells. So, what I'm gonna do is tell you about a few features of this organelle that I find really fascinating. And some of the... and I'll try and allude to those points where I think there's really critical knowledge about the mitochondria that we don't yet have, and that we need to understand better. And I will also tell you that
2:39
Jodi Nunnari gave an excellent talk about mitochondria that's part of the iBiology series, that talks about mitochondria primarily from the perspective of evolution and alludes to several features, and I definitely encourage you to watch that. And I'll try and cover some of the things that she didn't talk about quite so much. So, the five things... the five areas that I will allude to regarding mitochondria are shown here. First, about its origin.
3:07
It's clear, quite clear from how we understand mitochondria, that they evolved as a result of an aerobic bacterium becoming engulfed in what was a protoeukaryotic cell, and essentially became domesticated. Domesticated bacteria living in those cells that eventually entered into a symbiotic relationship that was of benefit both to that bacterium and that cell. And those bacteria have evolved and adapted and have become today's mitochondria. And when we think about that,
3:44
that we essentially have a domesticated bacterium living in most of our cells, I think that has big implications for how we think about cell biology. And I'll allude to that a little bit later, but that, I think, changes, in a way, the relationship between mitochondria and the nucleus, compared to other organelles, where their origin is quite different. So, the structure of mitochondria is one of the most unique features of this organelle. Unlike other organe... most other organelles
4:18
-- those in animal cells in particular -- mitochondria have two membrane compart... two membrane systems. There is an outer membrane that completely surrounds the mitochondria, and everything is contained within that outer membrane. That outer membrane turns out to be relatively porous to small molecules. This inner membrane, however, which encapsulates a protein compartment known as the matrix, shown in blue... this inner membrane is thought to be completely sealed and is impermeable to ions,
4:54
except through specific transport processes that are critically important for the function of mitochondria. This inner membrane is highly invaginated and leads to these folded structures depicted here as cristae, which will become important later. But this also... this folding also creates a scenario where the surface area of the inner membrane is quite a bit larger than the surface area of the outer membrane. And again, that becomes important because this inner membrane is the site
5:29
of much of the important work that is done in mitochondria. And the mitochondrial matrix, I just want to point out, is where a lot of the chemistry is done. So, the enzymes that we consider to be localized to mitochondria... the vast majority of them are localized to the mitochondrial matrix, which again is completely segregated from the cytosol by virtue of this two-membrane system, again the inner membrane being the dominant one for conveying the separation. So, where do the proteins come from
6:03
that perform the work in mitochondria? 99% of those proteins, roughly, are synthesized in the cytosol on cytosolic ribosomes and then imported into mitochondria. But interestingly, 1% of the proteins found in mitochondria are actually synthesized there, and they are encoded by the mitochondrial genome. So, again, another very unique feature, for animal cells, of mitochondria is the maintenance of this mitochondrial genome, which is a relic, we believe, of the ancestral bacterial genome
6:40
that was first brought into this protoeukaryotic cell. This mitochondrial genome only encodes 13 proteins, but those 13 proteins are critically important for the respiration functions of mitochondria. And those 13 proteins... all of them co-assemble with other nuclear-encoded cytosolically synthesized proteins into large protein complexes, which creates very interesting and important challenges of coordination that we'll talk about later. So, metabolism... metabolism is
7:15
easily the most famous function of mitochondria. That's what we think about typically when we think about mitochondrial function. This metabolic function of mitochondria can be broken down into several flavors of function. The most well known and most well studied, probably, is the catabolic function of mitochondria, the processes by which mitochondria consume the food that we eat and enable the production of ATP. And we're gonna go through that in a lot of detail.
7:48
But mitochondria also play very important biosynthetic functions, in building the molecules that our cells need for duplication and repair. And there are also very important functions of mitochondria in controlling redox balance, which I won't talk about in great detail but are clearly very important. So, this system by which mitochondria consume food and make ATP is truly amazing, truly incredibly important, especially in those cells that consume a lot of ATP,
8:22
like cardiomyocytes -- heart muscle cells -- and neurons, which have enormous ATP demand. The vast majority of that ATP comes from mitochondria. How does that work? This is just a brief summary of that from the perspective of a carbohydrate like glucose. When glucose is brought into the cell, it's converted to pyruvic acid in the cytosol, which generates a little bit of ATP -- two molecules of ATP. To fully capture the energy of glucose, however, that pyruvate is taken into the mitochondria,
8:54
carbons are extracted and released as CO2, electrons are extracted and conveyed to the electron transport chain, which enables very efficient ATP production, leading to a much... in an idealized setting, 38 molecules of ATP. That is the way to generate ATP highly efficiently. But as I mentioned, in that context the carbon is lost as CO2. So, if that carbon needs to be used to build something, like DNA or proteins or lipids, one can't fully oxidize glucose to carbo... to CO2.
9:33
And so that balance between anabolic and catabolic functions of mitochondria is one I'll come back to later, but it appears to be critically important. It's clearly important in normal physiology, and it appears to be critically important in disease. So, this is an overall summary of how this happens. So... of how ATP synthesis is managed by the mitochondria. So, how does this actually work? In very simplistic terms, one of the key features of this is that the energy of food
10:06
is used to enable the pumping of protons through this respiratory system, which we'll talk about in much more detail, from the mitochondrial matrix into the cristae space, the intermembrane space between those two membranes. And then, as those protons flow back down their gradient, the energy from that is captured to make ATP. And that is the process by which food energy is used to fool... fuel ATP production. And we'll talk about both the proton pumping and the ATP production aspects of that
10:41
in a little bit more detail. So, again, this is a zoom-in of this system. Again, food molecules are used to feed the TCA cycle, the citric acid cycle or Krebs cycle. The carbon is lost as CO2, but the electrons are extracted and passed to the electron transport chain, which then, in conjunction with pumping... with passing electrons, pumps protons from the matrix to the intermembrane space. And then as those protons then flow back down their gradient in an energetically favorable process,
11:17
that energy is captured -- very elegantly -- by this beautiful machine, the ATP synthase, and coupled to the synthesis of ATP. And again, we're gonna talk about each of those processes in more details. So, unlike combustion or an explosion, where the energy from a fuel is lost as heat or light, this process... the mitochondria capture that energy by virtue of it being conveyed in very small, very discrete steps, not unlike how a turbine -- a series of turbines --
11:55
captures water flowing downhill through gravity, or flowing through a dam via gravity. That energy is captured and converted into... and stored in a way that it can then be used to generate ATP that our cells will use to fuel their pro... their... the processes that they need to power. So, this is how mitochondria do it. It's by capturing that energy in very discrete processes. And it is done primarily through the pumping of protons. So, this is a depiction of the electron transport chain.
12:30
And again, this complex I, or NADH dehydrogenase, captures the electrons that have been held in the NAD cofactor pool and then conveys those electrons to ubiquinone, and in... and in the process of doing so, pumps protons from the matrix to the intermembrane space. The quinone pool passes those electrons again, to complex III, or cytochrome c reductase, which again pumps protons as those electrons are eventually then conveyed to cytochrome c,
13:06
which again passes its electrons to cytochrome c oxidase, which pumps protons in conjunction with electrons being carried to oxygen, and then enabling the formation of water. So, this process, this series of many, many electron transport reactions, is coupled to the pumping of protons. And this depiction is inappropriate in a way, because it belies the massive complexity of these complexes. So, I just want to focus on complex I. So, this is a structure of complex I
13:43
that was solved a few years ago. 44 different proteins are required for the function of this complex, make up this complex, which exists both in the... in the... mit... in the membrane, which is about here, the membrane piece of complex I, and then this matrix arm of this complex. And these are all required for the conveyance of electrons from NADH, through a series of cofactors that sequentially capture those electrons and pass them on down the chain, eventually reaching the quinone,
14:18
where they are deposited. That then takes them, as I said before, to the next complex in the chain. And that conveyance of electrons is coupled to the movement of specific helices in this membrane... in this membrane arm that enable the pumping of protons from the matrix to the intermembrane space. How that exactly works, we don't really know. We know some of the features of it, and we're trying... the field is trying very hard to understand this in more detail. But it's a very elegant process
14:54
by which these two functions of electron passage and proton pumping are coupled through this very complex, 44-subunit enzyme system. So, how does this proton gradient then get used to make ATP through ATP synthase? So, I want to show you an animation that I think illustrates this very nicely, based on actual structural data of this complex. So, the ATP synthase molecule is shown... or... complex is shown here. It's composed of two different motors
15:35
-- a motor in the matrix and one in the... in the membrane -- that use the proton gradient, as you see here. High in the matrix. You see low proton availability in the IMS. Those protons flow through this complex, and that flowing is coupled with ATP synthesis. And these two motors... the F0 motor that exists in the membrane captures those electrons and couples their flowing through the... to the rotation of this rotor. And the F1 motor makes ATP. So, protons come in through this rear channel,
16:14
bind to this F0 rotor, go all the way around, 360 degrees around this rotor, and then are released through this front channel into the matrix. And that powers the rotation of this F0 motor. That rotation is conveyed to the F1 motor through this central stalk, which rotates in the middle of this F1 motor and causes -- sequentially, as it goes around -- conformational changes that convey energy to the production of ATP from ADP and phosphate. And this goes around and around,
16:54
coupled with the pumping of protons, with the flowing of protons from the IMS... from the intermembrane space to the matrix, and coupling that with ATP synthesis. It's a really beautiful motor system to convey the potential energy of the proton gradient to the production of ATP that can be used for energetically demanding processes in the cell. I wanted to talk a little bit about the anabolic functions of metabolism... of the mitochondria as well. So, this is a complex slide,
17:29
and I just want to point out a few things. So, again, glucose comes into the cell. It's converted to pyruvate in the cytosol. That pyruvate is transported into the mitochondria and enters the TCA cycle. That TCA cycle not only extracts electrons to fuel the electron transport chain but also provides intermediates, like citrate, that can then be exported to the cytosol and are critically important for the synthesis of lipids, like fatty acids and cholesterol. So, mitochondria actually contribute
18:00
to the building of molecules as well as their destruction, like this... in this case with lipid synthesis. Oxaloacetate, another TCA cycle intermediate, is also used in the building of amino acids like aspartate, which contribute to nucleotide biosynthesis. So, again, very important biosynthetic functions of mitochondria, in addition to the catabolic, ATP-producing functions that we tend to focus on mostly. Interestingly, many pathways are actually shared
18:35
between the cytosol and the mitochondria, or the mitochondria and another organelle. Phospholipid biosynthesis is one example. One example is also shown here, which is particularly interesting, like this SHMT enzyme, which interconverts glycine and serine. There's actually an isoform in the cytosol and an isoform in the mitochondria that do the same reaction. And it's interesting to speculate that perhaps that enables the function of this pathway -- and others like it --
19:02
to exist... to be functional in different metabolic states, where the cytosol and the mitochondria might be differentially optimized for this chemistry in one condition versus another. Something that we really need to understand better is how the cytosolic and mitochondrial metabolic functions are coordinated. And in addition, one other type of coordination that is clearly important is the coordination between the anabolic functions of mitochondria, as shown here,
19:32
and the catabolic functions, which we just talked about. Clearly, those are both very important, and the mitochondria conducts all of them. How does it coordinate them? How does it decide whether it's more important to generate ATP or more important to generate building blocks for cell growth and proliferation and repair? It's something that we don't understand very well just yet, but it's something very critical for human... normal physiology and for disease.
19:59
So, I want to talk about the challenges that arise for protein homeostasis because of the unique structural constraints provided by mitochondria. So, this is, again, another depiction of the mitochondrial electron transport chain and the TCA cycle. And I just want to point out again that the electrons are extracted from acetyl-CoA, passed to this system through these very complex electron transport chain complexes... and eventually conveying the electrons to oxygen to make water,
20:35
while pumping protons. As I described, this is critically important to eventually enable ATP synthase to make ATP by using that proton gradient. A beautifully elegant system, fantastically complex. And that complexity brings several challenges that eventually end up being protein homeostasis challenges. So, just focusing on the... just the protein components of these complexes. So, just complex I, for example, as I alluded to, has 44 different subunits,
21:09
7 of which are encoded by the mitochondrial genome, 37 by the nuclear genome. And these proteins all have to find one another, in the right stoichiometry, to make an intact complex I. It's impossible for the cell to manage that in complete... with complete control. And the results have to be that too much of one subunit or too little of another is there. And that leads to a degradation problem, where those excess subunits have to be degraded. And this is happening throughout this complex.
21:40
That's an important problem that the mitochondria have to deal with. In addition to that, all of these complexes also have redox active cofactors that need to be managed very carefully. And when they're not managed carefully, that leads to the production of reactive oxygen species that create damage to the mitochondr... to mitochondrial proteins. Again, those proteins that are damaged have to be degraded, and recognized as being damaged, and degraded.
22:12
And... so, this is a massive protein homeostasis problem. And this is made worse by the fact that this is all happening in a place that is inaccessible to the proteasome, which does most of the protein degradation in the cell. So, that creates a challenge for how do proteins... how are proteins recognized and degraded in the mitochondria? And this is one depiction of the... the complexity of systems that do that. This system includes several proteases in the matrix, in the inner membrane,
22:46
and in the intermembrane space that, through mechanisms we mostly don't understand, recognize proteins that need to... need to be degraded and degrade them. There are also proteins on the outer membrane that need to be degraded, extracted from the outer membrane, and typically presented to the proteasome for degradation. How are they recognized as being aberrant is... still remains to be determined. And interestingly, it's becoming clear that the mitochondria is a common destination
23:15
for the mislocalization of proteins that should be somewhere else but end up mislocalizing to the mitochondrial outer membrane. How are they then recognized and degraded? It's a very important question that is clearly important for human disease. We really don't understand it very well. But sometimes, even all this complexity of systems trying to maintain mitochondrial quality doesn't work well enough, and mitochondria need to be degraded.
23:43
And there's a very interesting process known as mitochondrial autophagy, or mitophagy, that manages the degradation of whole organelles. This is mediated... one part... one pathway toward mitophagy is mediated by this PINK/Parkin system, which targets mitochondria for autophagy. Parkin is so named because mutations in the Parkin... the gene encoding Parkin cause, also, Parkinson's disease, a devastating neurodegenerative disorder, just emphasizing the importance of mitochondrial quality,
24:17
both in terms of protein degradation as well as mitochondrial degradation, for maintaining the normal function of healthy cells and healthy organisms. Finally, I want to just allude to some of the interesting communication that happens from mitochondria out to the rest of the cell, to the nucleus and the cytosol. There is also, of course, very interesting and important communication happening going in to mitochondria. I won't talk much about that.
24:50
But one of the obvious ways that mitochondria communicate with the rest of the cell is through providing metabolites, as we just talked about. And several of the metabolites that are produced by mitochondria have very important signaling and regulatory roles, like acetyl-CoA, alpha-ketoglutarate, ATP itself. They all play very important roles in controlling cell biology. And that's one of the key means whereby mitochondria communicate to the rest of the cell.
25:18
Another is the redox state of mitochondria. Because of its ability to put electrons into oxygen, this is... provides what is normally a very safe way to dispose of electrons. And when mitochondria don't have... when the cell doesn't have access to oxygen as an electron acceptor, through mitochondria, the cell has to adapt and find other ways to dispose of those electrons, or to not generate as many electrons. This is a very important part of mitochondrial function,
25:50
by controlling redox homeostasis, that we don't think about as much but is clearly important in several disease settings. We talk about reactive oxygen species, typically, as being signs of damage, when things go wrong. But it's clear that hydrogen peroxide and other oxidants play normal signaling roles, and we're just starting to understand the means whereby that signaling happens. What are the targets of that signaling?
26:17
And how does it control cell biology? But it's interesting to speculate that that might be a very interesting sentinel of how mitochondrial function is going, by virtue of the production of hydrogen peroxide. Calcium signaling is... mitochondria play a very important role in calcium signaling, both by virtue of their storing some calcium as well as controlling the release of calcium from other sites. And that... and obviously, calcium signaling is critically important in many excitable cells.
26:51
Mitochondria directly contact almost every other organelle, and probably we'll come to know that they contact every other membrane system in the cell. And by virtue of those contacts, that enables efficient transport of metabolites between organelles, and it's clear that there is also regulatory information conveyed by those interactions as well, and this is a very active area of research currently. One of, I think, the most interesting features of mitochondria
27:18
is their connection with the immune system. Again, this harkens back to the... to the history of mitochondria as a... as a bacterium that became engulfed by a eukaryotic cell. It's interesting to know, now, that many features of the immune system have particular relationships with mitochondria. Some systems of the innate immune response actually reside on mitochondria all the time, are centered there. And it's interesting to think about the implications of that, given its origin as a bacterium.
27:55
And there are very interesting responses to the exposure of mitochondrial DNA to the cytosol. And again, that's quite interesting, given the history of that mitochondrial genome as the descendant of what was once a bacterial genome. And the net effect of this is mitochondrial functions don't just make ATP. They don't just make metabolites. They significantly influence the behaviors that cells make. And those behaviors, when they become aberrant...
28:25
it's very clear have profound impacts on disease. And so, I think one of the most interesting areas of future research for us is to understand how mitochondria communicate, through these and other mechanisms, to control the behaviors of cells. And I'll talk in much more detail about that in the second part of this series. So, this leads us back to my story of why we became so interested in mitochondria. I think I told you about all the mysteries that I think
28:55
are still contained within this organelle. And this was brought home to me, quite clearly, by the elegant work of Dave Pagliarini and Vamsi Mootha when they defined the mitochondrial proteome in humans, several years back now. And it became clear that about a third of the proteins found there were completely uncharacterized. For this organelle that's been so well studied, about a third of the... about a third of the proteins found there have no known function. They likely are doing something.
29:26
And that likeliness that they're doing something is really illustrated by the fact that many of those proteins are found throughout all eukaryotes, implying conservation across many, many years of evolution. So, that function that they're doing is not important just for yeast, or just for humans, but throughout all eukaryotes. So, we decided in my lab to take a stab at understanding the functions of some of these uncharacterized, highly conserved mitochondrial proteins.
29:54
And initially... initially characterized that group, 32 protein families, that we set out to just go find the functions of, going one by one. And that's led us into many different areas of mitochondrial biology, some of which are shown here, along with the protein functions that we've identified. And I'll focus in Parts 2 and 3 of this series on two stories that emerged from this project, where we, by taking... by taking an approach to try and go into the mysterious features of mitochondria
30:27
and take what was unknown, and really try and understand the functions that those proteins have, that's led us into some very interesting of areas of mitochondrial biology. And I'll tell you more about that in Parts 2 and 3, and thank you for listening.
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