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Can proteins be located on the surface of the mitochondria?

Can proteins be located on the surface of the mitochondria?


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I am learning about the mitochondria and I know there are proteins present in the mitochondrial matrix such as SOD2, but I was wondering for a protein to be located on the surface of the mitochondria does it always have to be a membrane protein (e.g. inserted within or associated with the mitochondrial outer membrane) or can other proteins which are not membrane proteins be present on the mitochondrial surface? Any insights are appreciated.


Yes, integral membrane proteins interact with and form complexes with proteins that do not directly interact with the membrane.

Signal transduction often occurs when a soluble protein comes in from somewhere else and touches a membrane-bound protein.

Complexes often take the form of one anchored protein bound to additional proteins.

You can look at the full list of proteins associated with the mitochondrial membrane here, i bet if you go through them you will start to find ones that don't have a membrane-inserted domain.


Biology Tea

How do stains used for light microscopy compare with those used for electron microscopy?

Stains used for light microscopy can be described as colored molecules that bind to cell components, which alters the light passing through the specimen and then the glass lens. Stains for (transmission and scanning) electron microscopy involve heavy metals that affect the beams of electrons which are being propelled at the stain that either go through the specimen or onto its surface depending which microscope. These microscopes give higher resolutions, because the wavelengths of electrons are much shorter than those of visible light.

Which type of microscope would you use to study the changes in shape of a living white blood cell? Justify your response.

Since the objective is not to study any organelles, a light microscope will be suitable for this task. A light microscope is able to see mitochondria and if it is super-resolution, it can see ribosomes at the most. Therefore, the white blood cell size falls within the visible parameters of the light microscope.

Which type of microscope would you use to study the details of surface texture of a hair? Justify your response.

For this task, I would use a scanning electron microscope. The image given by using this type of microscope will appear to be something like a 3-D picture of the surface of the hair. An electron microscope would go straight through the hair, thus not allowing the scientist to see its surface. The sample is usually coated with gold for scanning microscopes and electrons are translated to a video screen.


Abstract

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C. albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the N A D H u biquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1. NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida-like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary “gain” of new amino acid inserts that are also the functional hub of the protein.

Citation: Mamouei Z, Singh S, Lemire B, Gu Y, Alqarihi A, Nabeela S, et al. (2021) An evolutionarily diverged mitochondrial protein controls biofilm growth and virulence in Candida albicans. PLoS Biol 19(3): e3000957. https://doi.org/10.1371/journal.pbio.3000957

Academic Editor: Anita Sil, University of California, San Francisco, UNITED STATES

Received: September 11, 2020 Accepted: January 29, 2021 Published: March 15, 2021

Copyright: © 2021 Mamouei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data can be found within the manuscript.

Funding: This work was supported by NIH grant R01AI141794-01A1 awarded to PU, 1R01AI141202-01 awarded to AI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: 3D, three-dimensional BN-PAGE, Blue native PAGE BSA, bovine serum albumin CFU, colony-forming unit CI, Complex I CII, Complex II DOX, doxycycline ETC, electron transport chain FDA, fluorescein diacetate FGSC, Fungal Genetics Stock Center FPS, farnesyl thiopyrophosphate HUVEC, human umbilical cord endothelial cell LDH, lactate dehydrogenase mNDU1, mature NDU1 MOPS, morpholinepropanesulfonic acid OCR, oxygen consumption rate OPK, opsonophagocytic killing OPM, Orientations of Proteins in Membranes PHYS, phytoene synthase PMSF, phenylmethylsulfonyl fluoride ROS, reactive oxygen species SE, silicone elastomer SQS, squalene synthase TCA, tricarboxylic acid WT, wild-type


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Part 2: Quality Control of Protein Localization

00:00:13.03 Hello.
00:00:14.03 My name is Manu Hegde.
00:00:15.03 I'm a group leader at the MRC Laboratory of Molecular Biology in Cambridge, England, and
00:00:20.10 today I'm going to talk to you about how protein localization is monitored for errors and failures.
00:00:26.09 Now, this is the second part of a three-set. of a series of three talks, and in the first
00:00:31.24 part I gave you a history of how the basic principles of protein localization were discovered.
00:00:38.18 So, for example, in a cell you have many different compartments -- peroxisomes, mitochondria,
00:00:44.13 ER, and so forth -- and what I discussed were the basic principles by which proteins are
00:00:50.11 selectively segregated among these different compartments.
00:00:53.22 And the idea is that, as proteins are being synthesized or shortly after their synthesis,
00:00:59.19 specific sequences within them are recognized, and those sequences are then recognized by
00:01:06.16 machinery that takes them to these different organelles.
00:01:09.18 Now, as nice as this system is, it turns out that the system does fail from time to time
00:01:16.00 not only can you have genetic mutations in a protein, for example, in the signal sequence,
00:01:20.14 that might cause it to not be translocated properly, but you might even have genetic
00:01:25.03 mutations in the machinery that mediates the targeting to these different organelles.
00:01:30.08 So, how then does the cell deal with these failures?
00:01:33.17 And, in fact, this wasn't a problem that was really considered through much of the part.
00:01:39.06 of the period when the machinery for targeting and translocation were being deciphered.
00:01:44.03 And so, what I'm going to tell you about is really my own personal experience with how
00:01:48.15 we came to realize that protein segregation to organelles might in fact be prone to failure
00:01:54.03 at a higher rate than we really appreciated, why we think this is important and what the
00:01:58.20 consequences are, and, most importantly, how the cell has evolved mechanisms to deal with
00:02:05.05 errors in protein localization.
00:02:08.07 So, the story begins when I was a graduate student at UCSF, and when I was at UCSF I
00:02:15.09 joined a lab, the lab of Vishu Lingappa, that was interested in how proteins get into the
00:02:20.24 endoplasmic reticulum.
00:02:22.14 Now, the ER. and a picture of the ER in a typical tissue culture cell is shown here.
00:02:28.21 is a vast organelle that's distr. that's distributed throughout the cell, and the types
00:02:33.23 of proteins that go into the ER are proteins that eventually have to reside on the membrane
00:02:39.10 of the cell surface or various intracellular membranes, or get secreted to the outside
00:02:44.08 of the cell.
00:02:45.13 And so what happens is that those proteins are synthesized by cytosolic ribosomes that
00:02:51.03 are selectively taken to the surface of the ER where the proteins are either imported
00:02:55.09 across the membrane or inserted into the membrane, before being trafficked to other parts of the cell.
00:03:01.19 And so the way this process historically was. was studied to try to understand the mechanisms
00:03:07.03 of how these events work is to try to reconstitute some of these events, such as the translocation
00:03:13.03 of a protein across the membrane, in a test-tube system.
00:03:17.16 And so the idea is that, if you could pull apart the components involved in such a process,
00:03:22.05 you might understand the machinery that makes it work.
00:03:24.23 And so I discussed some of the experiments that led to that. uhh. the basic principles
00:03:29.06 in the first part of the talk, and so this is the kind of experiment that we would do
00:03:33.11 in the lab when I was a student.
00:03:36.02 So, what this system contains is a cytosol that contains all the factors needed for protein synthesis.
00:03:43.21 We would add a radioactively labeled amino acid, which is. which is usually S-35 methionine,
00:03:50.12 and, if you want to study protein import, you would add a source of ER vesicles, which
00:03:55.21 is usually isolated from pancreas.
00:03:58.06 And you can manipulate these as well as many other components of the system
00:04:01.20 to try to dissect the processes.
00:04:03.21 So, of course, the process of protein translocation was historically studied with model proteins
00:04:10.01 that. that undergo translocation quite efficiently.
00:04:14.01 And so, one such model system that has been extensively studied is the hormone prolactin.
00:04:21.00 And so, what happens when you synthesize such a. such a protein in this in vitro lysate
00:04:26.04 is if you synthesize the protein without any ER membranes -- so that's shown all the way
00:04:31.02 on the left over here -- you get a product that is termed a precursor, and that's because,
00:04:36.18 without any place for that protein to go, it's synthesized and retained in the cytosol
00:04:41.23 where it still contains its signal peptide.
00:04:44.20 If, however, you have a complete reaction that has all the factors, then the protein
00:04:49.23 successfully gets imported into the ER, where that signal peptide gets removed and you get
00:04:55.14 a mature form of the protein.
00:04:57.21 So, that makes perfect sense, and you can of course show that that protein got into
00:05:02.01 the lumen of the ER because if you digest that sample with protease then you retain
00:05:08.14 protection of that product, and that's because it's protected inside the lumen of these vesicles.
00:05:13.18 And if you add protease in the presence of some detergent, which is this blank lane over
00:05:17.14 here, then everything is digested.
00:05:19.24 So, that looks great, and exactly this system was used to dissect the sequence of events
00:05:25.04 that lead to targeting and translocation of such a protein across the membrane.
00:05:30.04 Now, occasionally, you might want to look at non-model proteins.
00:05:34.20 Now, why would you do that?
00:05:36.03 Well, one of the main reasons you look at other proteins is if they are particularly
00:05:40.17 interesting for other reasons, such as their physiologic importance, or perhaps they're
00:05:45.15 involved in disease.
00:05:47.12 And so, when I was in Vishu's lab, one of the labs across the hall was Stan Prusiner's
00:05:51.18 lab, and the Prusiner lab was very interested in a set of neurodegenerative diseases called
00:05:56.18 prion diseases.
00:05:58.08 And this seemed to be a very fascinating set of diseases, somehow revolving around
00:06:02.15 protein misfolding.
00:06:04.00 So, Vishu at some point decided to try to look at what happens to prion protein when
00:06:09.02 it is first synthesized.
00:06:11.03 And it was thought to be entering the ER because it eventually goes to the surface of the cell,
00:06:16.23 and so we wondered, well, how does it normally fold when it first gets made?
00:06:22.10 And one gets a somewhat surprising result.
00:06:25.22 So, here's an experiment in which you synthesize prion protein and in the first lane you have
00:06:30.18 the complete reaction.
00:06:32.00 And, of course, as expected, the major product is the mature product, the one that corresponds
00:06:37.03 to the protein that gets across the membrane and has its signal peptide processed.
00:06:41.18 But, what you see is that there's a little bit of precursor that's left.
00:06:45.18 So, that was not quite seen with prolactin, which seemed to be a bit more efficient, so
00:06:50.18 what that really suggested was there was a little bit of inefficiency in which this precursor
00:06:55.18 was retained partially in the cytosol.
00:06:57.19 But, the slightly more surprising thing are these faint bands that are seen closer to
00:07:02.06 the bottom of the gel, and these were generated when you treated the sample with protease.
00:07:07.14 And what happens then is some of the protein gets digested to smaller fragments.
00:07:12.17 Now, it took a little bit of detective work to figure out exactly what these fragments
00:07:16.17 were, but they turn out to be transmembrane forms of the protein.
00:07:20.23 So, the reason they generate these fragments is that the cytosolic part of it gets digested
00:07:25.15 by protease, here and here, leaving the other parts protected from protease.
00:07:32.00 And because these protected fragments are slightly different sizes, you get two different
00:07:36.10 bands at the bottom of the gel.
00:07:38.06 So, what exactly was going on here?
00:07:40.14 And a little bit more detective work from various experiments that I did demonstrated
00:07:46.06 that what was happening is that there was this region of the protein that was slightly
00:07:52.05 hydrophobic, and so you look at this hydrophobic region and it's got a bunch of residues that
00:07:56.22 are moderately hydrophobic, like alanine, and some that are more hydrophobic,
00:08:01.00 like leucine or valine.
00:08:02.21 And it turns out that this sequence looks almost like a transmembrane segment.
00:08:08.10 And so what seemed to be happening is that, when you make it in this in vitro system,
00:08:13.00 the components in that system seem to fail to recognize that this protein should be translocated
00:08:19.04 across the membrane and a small amount of it gets inserted into the bilayer.
00:08:25.02 And so, at this point, these were just the initial experiments I did when I was first
00:08:29.21 starting in the lab, and I thought that it would be extremely interesting to figure out
00:08:34.13 how it is that these forms get generated, because it seemed very interesting.
00:08:38.15 It was different than the model protein prolactin and I wanted to not only figure out the sequence
00:08:43.06 of events that led to these. these transmembrane forms, but to identify factors that were involved
00:08:48.03 in making them.
00:08:49.11 So, this is what I proposed to do for my PhD, and when I then went and proposed this to
00:08:55.00 my qualifying exam committee they thought it was a terrible idea.
00:08:58.19 And, of course, they had a point, because after all these forms had only been seen in
00:09:04.00 this in vitro reaction.
00:09:05.08 The in vitro reaction, from a certain perspective, seems very strange because it's made of a
00:09:11.00 lysate from reticulocytes, membranes that come from pancreas. whereas the prion protein
00:09:16.10 is primarily expressed in neurons.
00:09:18.15 And so it was. it was deemed that these were almost certainly artifacts and I then
00:09:23.16 failed my qualifying exams.
00:09:25.13 So, what was I to do?
00:09:27.19 Well, the concern was that this was really irrelevant to anything biologically significant,
00:09:33.19 so of course I wanted to really see if that was the case or not.
00:09:37.23 And so the experiment then was, since we knew what it was that caused this to be made in
00:09:43.03 a transmembrane form, we could take that region of the protein and increase the hydrophobicity
00:09:48.17 of some of the amino acids -- for example, this alanine changed to a valine.
00:09:53.09 And then, to see if these forms were important at all, you could determine if these changes,
00:09:59.06 which in an in vitro system led to a little bit more of this transmembrane form, had any
00:10:03.20 consequence when you expressed it in transgenic mice.
00:10:07.13 And so we worked with the Prusiner lab, which had great mouse facilities, to express these
00:10:12.00 in mice and see what happens.
00:10:14.07 And, of course, during the few years that it took to wait for those mice to be made
00:10:19.03 and wait to see if they had any phenotype, I then went back to the lab and learned biochemistry
00:10:24.00 and actually did some work to try to figure out how these forms are being made.
00:10:28.23 So, the result was striking.
00:10:31.05 First of all, wild-type mice or mice that don't have prion protein at all live reasonably
00:10:36.10 long lives -- they live about two to three years.
00:10:39.12 But, when you express these mutations, for example this alanine to valine mutation or
00:10:44.17 this asparagine to isoleucine mutation, the mice got neurodegeneration and died at earlier
00:10:49.21 and earlier ages.
00:10:51.13 And the length of time they lived corresponded inversely to the amount of this transmembrane
00:10:57.00 form that was being generated based on our in vitro studies.
00:11:01.10 And if you looked in the brains of these mice, it appeared that they were developing this
00:11:05.12 type of spongiform neurodegeneration that had been seen in certain patients with very
00:11:11.07 similar mutations.
00:11:13.08 And so, what we can conclude from these studies is that the cell apparently -- or the brain
00:11:19.05 -- tolerates a little this transmembrane form, and when we did careful measurements we could
00:11:23.19 detect a couple percent made in this particular transmembrane form, but mutations that result
00:11:30.04 in a little bit more, for example instead of 2%, 5%, is sufficient to cause neurodegeneration.
00:11:37.16 And that was true not only in mice but it turned out that in that central hydrophobic
00:11:41.21 region, there were a number of families that had mutations that were very similar to the
00:11:46.15 artificial ones we had made, in which residues such as alanine or glycine were changed to
00:11:51.13 more hydrophobic residues.
00:11:53.09 So, it appeared, in fact, that mislocalizing the protein into the membrane when it was
00:11:58.19 not supposed to be in the membrane can be detrimental, and that then made us quite a
00:12:03.21 bit more interested in how this form was actually generated.
00:12:08.11 So, of course, I then left the lab and started my own lab, and in the beginning parts of
00:12:15.03 our studies we then tried to investigate how this transmembrane form, here, gets generated
00:12:20.20 when you start with a protein that's just being synthesized.
00:12:24.04 So, we wanted to fill in this gap between when you start synthesis and how you get this
00:12:28.20 transmembrane form.
00:12:30.10 And so, Soo Jung Kim, who was the first postdoc to join my lab, took on this project, and
00:12:35.24 what Soo found was that after you initially start making your protein that signal peptide
00:12:41.18 that's on the prion protein gets recognized by the signal recognition particle, SRP, and
00:12:45.19 SRP has a receptor at the surf. at the surface of the ER called SRP receptor, and then that
00:12:53.18 protein gets transferred to the translocation channel.
00:12:57.03 So, to this point, everything looks exactly the same as it does for any model protein,
00:13:01.24 and this is what happens to pretty much all proteins that are supposed to be imported
00:13:05.15 in the lumen.
00:13:06.15 And, in fact, the next step would be that this signal would engage the Sec61 channel,
00:13:11.16 open that channel, and the protein would go across the membrane.
00:13:14.20 So, what was happening here?
00:13:17.09 It turned out that when we looked carefully, the interaction between the signal and the
00:13:22.02 translocon was a bit more dynamic than, for example, the model protein prolactin, and
00:13:28.01 so about 10-20% of the time the signal peptide wouldn't engage quite properly, and then,
00:13:35.01 because all of these events here are occurring cotranslationally, the protein continues to
00:13:40.05 be synthesized and this hydrophobic region, which is in red, gets synthesized and starts
00:13:45.18 coming out of the ribosome.
00:13:47.19 And when that happens, now you have a hydrophobic region that looks a little bit like a transmembrane
00:13:52.14 segment, right next to the translocation channel, which is designed to recognize
00:13:57.14 transmembrane segments.
00:13:59.09 So, a small amount of the time, that transmembrane-like region inserts in the membrane via Sec61.
00:14:06.09 The rest of the time, these failed products are just released into the cytosol and that
00:14:12.04 cytosolic product is degraded.
00:14:15.04 And so this provided, then, a sequence of events for how you could generate
00:14:19.18 this transmembrane form.
00:14:21.16 But, of course, it was a bit puzzling because. why would you have a protein whose signal
00:14:26.23 peptide was not ideally efficient like the prolactin signal was?
00:14:31.03 And what was even more puzzling is when we looked at the signal peptide of prion protein
00:14:35.08 from different species, for example, bovine or mice, rats, humans, they were always slightly
00:14:41.17 inefficient to about the same degree.
00:14:44.03 And so it was unclear why this should be the case and we suspected that maybe there were
00:14:48.04 certain conditions in which having this type of a signal was beneficial.
00:14:53.07 And so Sang-Wook Kang in the lab decided to investigate this, and what Sang found, and
00:15:00.05 he was working with Neena Rane, another postdoc in the lab, is that this dynamic interaction
00:15:06.03 is, under normal conditions, resulting in about 80-90% of the protein entering the ER,
00:15:12.10 and a little bit released to the cytosol.
00:15:14.17 But, under acute ER stress conditions -- and what ER stress is is when the folding capacity
00:15:20.17 of the lumen of the ER is compromised -- then the situation was different.
00:15:25.00 And now, a much higher proportion of the protein was rejected and less of it gets into the
00:15:31.15 lumen of this stressed ER.
00:15:34.03 And it turns out that this is beneficial for the cell.
00:15:36.24 And the way we know this is because if you replace the signal peptide of prion protein
00:15:41.05 with a much more efficient signal peptide, then the protein is forced to go into the
00:15:45.18 ER and, under conditions of acute ER stress, winds up aggregating in the lumen of the ER,
00:15:51.20 and that turns out to be detrimental.
00:15:53.10 So, this provided a reasonably satisfactory explanation for at least one situation where
00:15:59.04 having this type of a signal is beneficial.
00:16:02.08 And, of course, proteins like prolactin are constitutively translocated even under conditions
00:16:09.04 of ER stress.
00:16:12.05 So, what then happens when you have chronic mistranslocation?
00:16:18.08 And the reason we were interested in this is because we showed that under ER stress
00:16:23.18 about 50% of the protein gets rejected, and it's been observed that under various disease
00:16:29.17 conditions cells experience various types of stress, including ER stress.
00:16:35.02 And so we wondered whether chronic mistranslocation, that is, chronic failure to import about half
00:16:41.01 the protein, has any consequences.
00:16:43.24 And what Neena Rane found, when she made a transgenic mouse that expresses a version
00:16:47.20 of prion protein with a partially inefficient signal peptide, is that that mouse, although
00:16:52.23 it lives a roughly normal lifespan, develops neurodegeneration over time.
00:16:58.01 And so you can see, I hope, that this mouse is a bit ataxic, meaning it doesn't quite
00:17:02.16 have proper balance and. and wobbles a bit, it has this hunched back and it doesn't groom
00:17:08.17 properly, and it has various signs of neurodegeneration.
00:17:12.09 And so, what we can conclude is that, just like this transmembrane form where a little
00:17:17.12 bit is tolerated but a slight excess is not tolerated, the same thing holds for
00:17:22.24 this cytosolic form.
00:17:24.17 If everything is normal, it's reasonably well tolerated, and a higher amount may be tolerated
00:17:31.03 for short periods of time, for example, during acute ER stress, but over long periods this
00:17:36.12 also is not tolerated.
00:17:38.14 And this. these are effects that you see in an intact organism over long periods of
00:17:43.04 time, so, in a cultured cell, these effects are very subtle if. and so are very hard
00:17:47.24 to detect, if detectable at all.
00:17:50.09 And so, what this suggested, then, is that these forms are mislocalized, and these mislocalized
00:17:58.22 forms of the protein are somehow detrimental.
00:18:01.23 Now, this is where having some understanding of how these forms are generated is useful,
00:18:07.22 because what we knew was that both this transmembrane form and this cytosolic form completely rely
00:18:14.14 on having a signal peptide that is slightly inefficient in the way it engages
00:18:19.10 the Sec61 translocon.
00:18:21.14 And that makes a very specific prediction, which is that both of these forms should be
00:18:27.08 avoidable if you increase the efficiency of the signal peptide at this earlier step.
00:18:33.24 And so, what that means is that you should be able to take a mutation that normally would
00:18:38.06 generate higher amounts of this transmembrane form and cause disease and rescue it by changing
00:18:44.12 the signal peptide.
00:18:46.03 And we already knew that some signal peptides, like prolactin, are much more efficient and,
00:18:50.20 in fact, we had confirmed in vitro that if you put the prolactin signal onto prion protein
00:18:56.06 you could reduce generation of these forms of PrP.
00:18:59.23 So, Neena did that experiment and the result was remarkable, because it basically showed
00:19:06.19 that, in fact, in vivo, in a mouse, the prion protein signal must be slightly inefficient.
00:19:13.05 And so what you can see here is. here's a mouse with this alanine to valine mutation
00:19:17.19 and. and that's the one that's basically just sitting here not doing much, and a matched
00:19:23.22 mouse in which it still has that mutation but now has a more efficient signal.
00:19:30.03 And that mouse turns out to be much healthier, it lives a bit longer, and doesn't develop
00:19:35.24 neurodegeneration, whereas this alanine to valine mouse over here develops neurodegeneration.
00:19:40.20 And we were able to get this type of a rescue with two different efficient signal peptides,
00:19:46.02 and with two different disease-causing mutations.
00:19:48.22 So, we were quite certain that, under normal conditions, prion protein in fact must have
00:19:55.02 a slightly inefficient signal peptide, even in an intact living animal.
00:20:01.24 And that really put to rest this notion that inefficiencies in the signal peptide or these
00:20:08.04 minor aberrant forms were just artifacts of an in vitro system.
00:20:13.07 And so, what we learned from all of these experiments is that, in fact, errors during
00:20:18.11 biosynthesis seem to be pervasive -- because many of these things that I told you about
00:20:23.17 applied not only to prion protein signal peptide, but also to other signals -- and that subtle
00:20:28.12 excesses in these errors can lead to disease over time.
00:20:32.12 And, in fact, if you look in the human population, there are rare diseases in which signal peptides
00:20:38.16 of other types of proteins are mutated, and those often cause a dominant disease in the
00:20:45.06 tissue where that protein is expressed.
00:20:47.11 So, as an example, there are mutations in the signal peptide of insulin and those people
00:20:53.00 develop failure of the cells that. that are. that are producing insulin, the beta
00:20:57.19 cells of the pancreas.
00:20:59.13 And so, it suggests that failures in localization to the correct compartment lead to synthesis
00:21:07.07 of proteins which, when there mislocalized, can be detrimental over long periods of time.
00:21:12.24 And so, that then really raised the question of, how does the cell normally get rid of
00:21:17.16 these products?
00:21:19.05 And this is something that then we became much more interested in, having realized what
00:21:23.13 the physiologic significance of these failures are, and knowing that, even under normal conditions,
00:21:30.01 there's always some products that fail.
00:21:32.09 So, how do we approach this problem?
00:21:35.11 And here we again turned to a biochemical system because what we asked was, does this
00:21:41.09 process, a failure and degradation, work in a test tube?
00:21:45.20 Because if it does then we might be able to pick apart the components that are in this
00:21:49.13 test tube to try to identify factors involved in it.
00:21:53.01 And it turns out that the degradation doesn't work in our usual in-vitro translation system.
00:21:58.18 But, what you see is that if you synthesize in this experiment a prion protein without
00:22:04.18 ER vesicles, so that all of it is made in this mislocalized form, you generate this
00:22:09.13 precursor which is not degraded, but that then does give you these extra bands.
00:22:17.16 In the complete reaction, the protein is imported into the lumen of the ER where it gets glycosylated
00:22:23.23 and the signal peptide gets removed.
00:22:25.22 So, what are these extra bands.
00:22:28.04 And these extra bands turn out to be attachment of a small protein called ubiquitin, shown
00:22:33.20 here in red, to the prion protein.
00:22:36.17 And that was significant because, even though our protein was not getting degraded, it was
00:22:41.03 getting marked with this ubiquitin, which is a very well-known tag for degradation of
00:22:46.11 proteins in the cytosol.
00:22:47.22 So, proteins that are polyubiquitinated in this particular way are targeted to the proteasome,
00:22:53.15 which is a major degradation machinery in the cytosol, for degradation.
00:22:58.06 So, we reasoned that, even though degradation is not occurring, the part that we're really
00:23:03.11 interested in, which is recognition of this as an aberrant product that needs to be marked
00:23:08.19 for degradation. that step was occurring reasonably well in this in vitro system.
00:23:13.18 So, we could then ask, what is it that's recognizing these products to mark them with ubiquitin?
00:23:20.16 So, how can we do this?
00:23:22.21 And what we imagined is that there should be some recognition factor that identifies this.
00:23:30.09 And one of the features of this that we realized early on that was being recognized is the
00:23:35.09 uncleaved signal peptide.
00:23:38.04 And so we looked for factors that might recognize this product that still contains an uncleaved
00:23:43.19 signal peptide.
00:23:45.05 And there are lots of ways to do this because we know that our protein, here, is radioactive,
00:23:49.23 and so we can then, for example, see what the native molecular weight of our protein
00:23:55.12 is, because we know that the protein itself is a certain molecular weight, but if it behaves
00:24:00.09 as if it's bigger that suggests it might be associated with some recognition factor.
00:24:05.08 And, if that's the case, then you can try to make guesses as to what that might be either
00:24:11.23 by identifying it directly or by knowing some properties about it, for example the molecular
00:24:17.07 weight of the recognition factor.
00:24:19.24 And the way we can do this is you can take such a sample and you can treat it with a
00:24:24.10 crosslinker and a crosslinker will chemically link these two proteins together because they're
00:24:30.15 very close to each other.
00:24:31.24 So, it does so via reacting with certain amino acid side chains.
00:24:37.05 So, in this particular experiment, here's our translation product before crosslinking
00:24:41.21 where almost all of it is here at the bottom of the gel, and then after crosslinking you
00:24:46.20 can see that it's diminished, and some of it has shifted to these different sizes.
00:24:52.19 And what we knew was that the factor we were looking for was likely to be a very large
00:24:58.03 factor, because the activity for attaching ubiquitin to our protein was a large factor.
00:25:06.05 And so we focused, then, on this particularly large crosslinked product and identified
00:25:11.17 what it was.
00:25:13.03 And that product turns out to be a protein called Bag6.
00:25:16.07 And Bag6, it turns out, is itself part of a three-protein complex and its function really
00:25:23.01 wasn't very well understood.
00:25:24.23 But, what we could show was that Bag6 recognizes these hydrophobic sequences and, in fact,
00:25:31.05 it recognizes many unrelated mislocalized proteins.
00:25:34.20 And the way it seems to do so is it recognizes the parts of the protein that should have
00:25:39.13 been recognized by a different factor -- in this case, for example, SRP.
00:25:44.20 And so the idea, then, is that, under normal conditions, as a protein is being synthesized,
00:25:51.17 it will be recognized by SRP on the ribosome, taken to the membrane and recognized there
00:25:57.20 by Sec61, and then imported.
00:26:00.15 And because these events both occur on the ribosome itself, and SRP and Sec61 bind to
00:26:07.12 the ribosome, that I think explains why Bag6 doesn't interfere with these processes.
00:26:13.22 In essence, while translation is occurring, Bag6 really doesn't have access to these proteins.
00:26:20.12 And other proteins also. many of them are also made cotranslationally, and it seems
00:26:28.12 that these. these segments of the protein, these very hydrophobic regions, during biosynthesis
00:26:35.01 are either shielded by factors, and after biosynthesis are shielded inside the membrane.
00:26:41.16 And those are the sequences that Bag6 recognizes.
00:26:44.09 So, it seems that that's how Bag6 knows that a protein is mislocalized, because a region
00:26:50.01 of the protein that should be shielded is now exposed.
00:26:54.06 And so idea, then, is that under normal conditions the biosynthetic machinery -- SRP and Sec61
00:27:01.13 -- have priority, and that's because they're associated with the ribosome, and Bag6 doesn't
00:27:07.00 seem to interfere with that process.
00:27:09.04 But, when targeting fails, Bag6 then specifically recognizes these exposed signals and transmembrane
00:27:16.15 segments, and it does a few things.
00:27:19.19 First of all, it keeps these substrates shielded, and that's important because these hydrophobic
00:27:24.13 sequences can make a lot of promiscuous and nonspecific interactions,
00:27:28.20 and they can also aggregate.
00:27:31.07 So, having something that shields them is quite important to prevent aggregation.
00:27:35.23 And it recruits a ubiquitin ligase, and this is a type of enzyme that attaches ubiquitin
00:27:41.20 to our substrate, here, which is the red. the red bit, here.
00:27:45.11 And that would target the substrate for degradation.
00:27:48.05 So, Bag6 then is a factor that identifies mislocalized proteins and
00:27:53.14 targets them for degradation.
00:27:55.21 Now, the situation isn't quite that simple, because it turns out that, in addition to
00:28:00.08 this cotranslational pathway, there are other pathways that work after the protein is synthesized,
00:28:07.00 and certain types of proteins such as tail-anchored membrane proteins are targeted posttranslationally,
00:28:13.16 so I won't go into it but it turns out that the machinery for targeting these tail-anchored
00:28:18.16 proteins also gets to have priority before Bag6 acts.
00:28:23.21 And so, in both cases, Bag6 seems to be a factor that waits until you get a chance to
00:28:29.12 target properly to the right place, but is waiting to catch you if you fail and therefore
00:28:35.10 mark you for degradation.
00:28:37.02 And so, if you want to understand more details of exactly how this decision making works
00:28:41.17 in this more complicated posttranslational pathway, you can read the reference that's
00:28:46.03 cited here at the bottom.
00:28:48.22 So, ultimately then, we find that targeting to the ER is prone to occasional failures
00:28:54.24 and the cell seems to have evolved a mechanism to deal with those failures by having evolved
00:29:00.09 a specific factor that recognizes these mislocalized proteins and targets them for degradation.
00:29:06.01 And this discovery really inspired us to then look for other types of failures, for example,
00:29:12.24 proteins also go to mitochondria, and, when we looked, Eisuke Itakura in the lab found
00:29:18.17 a set of factors called ubiquilins that seemed to recognize membrane proteins destined for
00:29:24.12 mitochondria and targets them for degradation.
00:29:27.18 And, similarly, proteins also have to go to other organelles, for example, the nucleus.
00:29:32.23 So, all ribosomal proteins first are synthesized in the cytosol and go to the nucleus, where
00:29:39.13 they're assembled into ribosomes, and if that import fails. umm.
00:29:43.22 Kota Yanagitani has identified a factor that seems to recognize these mislocalized ribosomal
00:29:50.10 proteins and targets them for degradation.
00:29:52.16 And so the idea here is that the cell has evolved an entire set of factors that deals
00:29:57.23 with different types of failures in getting proteins to the right part of the cell.
00:30:02.14 And so it's apparent that, both under normal conditions, as well as certain pathologic
00:30:07.22 conditions, failure to get proteins to the right compartment is a problem that the cell
00:30:12.14 needs to deal with.
00:30:14.10 Not only that, but these factors themselves may become impaired during disease.
00:30:19.06 So, for example, the ubiquilins might be a particularly important example because there
00:30:24.16 are genetic mutations in ubiquilins that are found in certain rare instances of
00:30:30.13 neurodegenerative disease.
00:30:32.10 And, in addition, certain other cases of neurodegeneration seem to deplete ubiquilins from cells.
00:30:39.03 So, in this example, which is an experiment done by Eszter Zavodszky in my lab, she expressed
00:30:46.00 a mutant protein from the protein huntingtin, and this is a protein which, when mutated
00:30:51.21 in a certain way, causes Huntington's disease.
00:30:55.12 And what you can see is that the mutant huntingtin, which is red, winds up sequestering almost
00:31:01.17 all of the ubiquilin in these two cells, here.
00:31:04.23 And so, the ubiquilin, then, is no longer available in its normal part of the cell,
00:31:09.06 which is typically to be diffuse throughout the cell where it's monitoring that cell for
00:31:13.09 failures in mitochondrial targeting.
00:31:15.20 And, sure enough, if you look specifically in these cells that contain the aggregates,
00:31:21.16 the ubiquilin in those cells is deficient in serving its normal function.
00:31:26.17 And so what we think, then, is that the lack of this important quality control and housekeeping
00:31:32.22 function might be a contributing factor to why cells that have certain kinds of aggregates
00:31:39.02 are prone to death, and prone to eventual dysfunction.
00:31:44.21 So, what I then want to leave you with is that the process of protein localization to
00:31:51.01 different organelles, while quite sophisticated in the machinery that carries out this.
00:31:56.12 these. these events, is nevertheless prone to failure.
00:31:59.16 And those failures might be exaggerated during, for example, certain conditions like ER stress
00:32:04.24 or mitochondrial stress.
00:32:07.20 It's a normal housekeeping function for the cell to have evolved ways of dealing with
00:32:12.16 failures in this localization.
00:32:14.12 So, what are the challenges here?
00:32:17.02 And I think many of these factors have only just been discovered, and I think that it's
00:32:21.16 very unlikely we know all of the factors that deal with failures in different types of localization,
00:32:27.13 so it's quite important to get a complete parts list of all the factors involved.
00:32:31.21 The second thing is that, even though we've roughly matched up proteins that go to the
00:32:36.19 ER with Bag6, or proteins that fail to go to mitochondria with ubiquilins, I think that
00:32:42.10 we have a rather poor overall understanding of which quality control pathways are for
00:32:48.12 which clients.
00:32:49.14 And, related to that, I think we have a very poor idea of how recognition actually occurs.
00:32:56.14 And so we have a rough idea with Bag6 that it recognizes hydrophobic regions, but, overall,
00:33:03.10 the details of that recognition are unclear.
00:33:06.01 And finally, as I had told you at the end, we think that many of these pathways may be
00:33:09.22 impaired in disease and it's a major goal to try to understand exactly how they're impaired,
00:33:16.02 and whether anything can be done to either increase their efficacy or otherwise manipulate
00:33:21.22 the process in order to at least have some impact on these diseases.
00:33:25.17 So, let me end by pointing out that I've had the pleasure of working with a really great
00:33:32.13 group of people over the past seventeen years or so, some of them are shown here, and I've
00:33:37.17 tried to name the individuals that have carried out some of the key experiments as I've gone
00:33:42.23 through the talk.
00:33:44.09 The current group, in this picture that was taken last summer, is shown here.
00:33:49.02 Thank you for listening.


Contents

In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. Blobel, then an assistant professor at Rockefeller University, built upon the work of his colleague George Palade. [5] Palade had previously demonstrated that non-secreted proteins were translated by free ribosomes in the cytosol, while secreted proteins (and target proteins, in general) were translated by ribosomes bound to the endoplasmic reticulum. [5] Candidate explanations at the time postulated a processing difference between free and ER-bound ribosomes, but Blobel hypothesized that protein targeting relied on characteristics inherent to the proteins, rather than a difference in ribosomes. Supporting his hypothesis, Blobel discovered that many proteins have a short amino acid sequence at one end that functions like a postal code specifying an intracellular or extracellular destination. [2] He described these short sequences (generally 13 to 36 amino acids residues) [1] as signal peptides or signal sequences and was awarded the 1999 Nobel prize in Physiology for his findings. [6]

Signal peptides serve as targeting signals, enabling cellular transport machinery to direct proteins to specific intracellular or extracellular locations. While no consensus sequence has been identified for signal peptides, many nonetheless possess a characteristic tripartite structure: [1]

  1. A positively charged, hydrophilic region near the N-terminal.
  2. A span of 10 to 15 hydrophobic amino acids near the middle of the signal peptide.
  3. A slightly polar region near the C-terminal, typically favoring amino acids with smaller side chains at positions approaching the cleavage site.

After a protein has reached its destination, the signal peptide is generally cleaved by a signal peptidase. [1] Consequently, most mature proteins do not contain signal peptides. While most signal peptides are found at the N-terminal, in peroxisomes the targeting sequence is located on the C-terminal extension. [7] Unlike signal peptides, signal patches are composed by amino acid residues that are discontinuous in the primary sequence but become functional when folding brings them together on the protein surface. [8] Unlike most signal sequences, signal patches are not cleaved after sorting is complete. [9] In addition to intrinsic signaling sequences, protein modifications like glycosylations can also induce targeting to specific intracellular or extra cellular regions.

Since the translation of mRNA into protein by a ribosome takes place within the cytosol, proteins destined for secretion or a specific organelle must be translocated. [10] This process can occur during translation, known as co-translational translocation, or after translation is complete, known as post-translational translocation. [11]

Co-translational translocation Edit

Most secretory and membrane-bound proteins are co-translationally translocated. Proteins that reside in the endoplasmic reticulum (ER), golgi or endosomes also use the co-translational translocation pathway. This process begins while the protein is being synthesized on the ribosome, when a signal recognition particle (SRP) recognizes an N-terminal signal peptide of the nascent protein. [12] Binding of the SRP temporarily pauses synthesis while the ribosome-protein complex is transferred to an SRP receptor on the ER in eukaryotes, and the plasma membrane in prokaryotes. [13] There, the nascent protein is inserted into the translocon, a membrane-bound protein conducting channel composed of the Sec61 translocation complex in eukaryotes, and the homologous SecYEG complex in prokaryotes. [14] In secretory proteins and type I transmembrane proteins, the signal sequence is immediately cleaved from the nascent polypeptide once it has been translocated into the membrane of the ER (eukaryotes) or plasma membrane (prokaryotes) by signal peptidase. The signal sequence of type II membrane proteins and some polytopic membrane proteins are not cleaved off and therefore are referred to as signal anchor sequences. Within the ER, the protein is first covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. Once folded, the protein is modified as needed (for example, by glycosylation), then transported to the Golgi for further processing and goes to its target organelles or is retained in the ER by various ER retention mechanisms.

The amino acid chain of transmembrane proteins, which often are transmembrane receptors, passes through a membrane one or several times. These proteins are inserted into the membrane by translocation, until the process is interrupted by a stop-transfer sequence, also called a membrane anchor or signal-anchor sequence. [15] These complex membrane proteins are currently characterized using the same model of targeting that has been developed for secretory proteins. However, many complex multi-transmembrane proteins contain structural aspects that do not fit this model. Seven transmembrane G-protein coupled receptors (which represent about 5% of the genes in humans) mostly do not have an amino-terminal signal sequence. In contrast to secretory proteins, the first transmembrane domain acts as the first signal sequence, which targets them to the ER membrane. This also results in the translocation of the amino terminus of the protein into the ER membrane lumen. This translocation, which has been demonstrated with opsin with in vitro experiments, [16] [17] breaks the usual pattern of "co-translational" translocation which has always held for mammalian proteins targeted to the ER. A great deal of the mechanics of transmembrane topology and folding remains to be elucidated.

Post-translational translocation Edit

Even though most secretory proteins are co-translationally translocated, some are translated in the cytosol and later transported to the ER/plasma membrane by a post-translational system. In prokaryotes this process requires certain cofactors such as SecA and SecB and is facilitated by Sec62 and Sec63, two membrane-bound proteins. [18] The Sec63 complex, which is embedded in the ER membrane, causes hydrolysis of ATP, allowing chaperone proteins to bind to an exposed peptide chain and slide the polypeptide into the ER lumen. Once in the lumen the polypeptide chain can be folded properly. This process only occurs in unfolded proteins located in the cytosol. [19]

In addition, proteins targeted to other cellular destinations, such as mitochondria, chloroplasts, or peroxisomes, use specialized post-translational pathways. Proteins targeted for the nucleus are also translocated post-translationally through the addition of a nuclear localization signal (NLS) that promotes passage through the nuclear envelope via nuclear pores. [20]

Mitochondria Edit

Most mitochondrial proteins are synthesized as cytosolic precursors containing uptake peptide signals. Cytosolic chaperones deliver preproteins to channel-linked receptors in the mitochondrial membrane. The preprotein with presequence targeted for the mitochondria is bound by receptors and the general import pore (GIP), collectively known as translocase of the outer membrane (TOM), at the outer membrane. It is then translocated through TOM as hairpin loops. The preprotein is transported through the intermembrane space by small TIMs (which also acts as molecular chaperones) to the TIM23 or TIM22 (translocase of the inner membrane) at the inner membrane. Within the matrix the targeting sequence is cleaved off by mtHsp70.

Three mitochondrial outer membrane receptors are known:

  1. TOM70: Binds to internal targeting peptides and acts as a docking point for cytosolic chaperones.
  2. TOM20: Binds presequences.
  3. TOM22: Binds both presequences and internal targeting peptides.

The TOM channel (TOM40) is a cation specific high conductance channel with a molecular weight of 410 kDa and a pore diameter of 21Å.

The presequence translocase23 (TIM23) is localized to the mitochondrial inner membrane and acts as a pore-forming protein which binds precursor proteins with its N-terminus. TIM23 acts as a translocator for preproteins for the mitochondrial matrix, the inner mitochondrial membrane as well as for the intermembrane space. TIM50 is bound to TIM23 at the inner mitochondrial side and found to bind presequences. TIM44 is bound on the matrix side and found binding to mtHsp70.
The presequence translocase22 (TIM22) binds preproteins exclusively bound for the inner mitochondrial membrane.

Mitochondrial matrix targeting sequences are rich in positively charged amino acids and hydroxylated ones.

Proteins are targeted to submitochondrial compartments by multiple signals and several pathways.

Targeting to the outer membrane, intermembrane space, and inner membrane often requires another signal sequence in addition to the matrix targeting sequence.

Chloroplasts Edit

The preprotein for chloroplasts may contain a stromal import sequence or a stromal and thylakoid targeting sequence. The majority of preproteins are translocated through the Toc and Tic complexes located within the chloroplast envelope. In the stroma the stromal import sequence is cleaved off and folded as well as intra-chloroplast sorting to thylakoids continues. Proteins targeted to the envelope of chloroplasts usually lack cleavable sorting sequence.

Both chloroplasts and mitochondria Edit

Many proteins are needed in both mitochondria and chloroplasts. [21] In general the dual-targeting peptide is of intermediate character to the two specific ones. The targeting peptides of these proteins have a high content of basic and hydrophobic amino acids, a low content of negatively charged amino acids. They have a lower content of alanine and a higher content of leucine and phenylalanine. The dual targeted proteins have a more hydrophobic targeting peptide than both mitochondrial and chloroplastic ones. However, it is tedious to predict if a peptide is dual-targeted or not based on its physico-chemical characteristics.

Peroxisomes Edit

All peroxisomal proteins are encoded by nuclear genes. [22] To date there are two types of known Peroxisome Targeting Signals (PTS): [23]

  1. Peroxisome targeting signal 1 (PTS1): a C-terminal tripeptide with a consensus sequence (S/A/C)-(K/R/H)-(L/A). The most common PTS1 is serine-lysine-leucine (SKL). Most peroxisomal matrix proteins possess a PTS1 type signal.
  2. Peroxisome targeting signal 2 (PTS2): a nonapeptide located near the N-terminus with a consensus sequence (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F) (where X can be any amino acid).

There are also proteins that possess neither of these signals. Their transport may be based on a so-called "piggy-back" mechanism: such proteins associate with PTS1-possessing matrix proteins and are translocated into the peroxisomal matrix together with them. [24]

Protein transport is defective in the following genetic diseases:

As discussed above (see protein translocation), most prokaryotic membrane-bound and secretory proteins are targeted to the plasma membrane by either a co-translation pathway that uses bacterial SRP or a post-translation pathway that requires SecA and SecB. At the plasma membrane, these two pathways deliver proteins to the SecYEG translocon for translocation. Bacteria may have a single plasma membrane (Gram-positive bacteria), or an inner membrane plus an outer membrane separated by the periplasm (Gram-negative bacteria). Besides the plasma membrane the majority of prokaryotes lack membrane-bound organelles as found in eukaryotes, but they may assemble proteins onto various types of inclusions such as gas vesicles and storage granules.

Gram-negative bacteria Edit

In gram-negative bacteria proteins may be incorporated into the plasma membrane, the outer membrane, the periplasm or secreted into the environment. Systems for secreting proteins across the bacterial outer membrane may be quite complex and play key roles in pathogenesis. These systems may be described as type I secretion, type II secretion, etc.

Gram-positive bacteria Edit

In most gram-positive bacteria, certain proteins are targeted for export across the plasma membrane and subsequent covalent attachment to the bacterial cell wall. A specialized enzyme, sortase, cleaves the target protein at a characteristic recognition site near the protein C-terminus, such as an LPXTG motif (where X can be any amino acid), then transfers the protein onto the cell wall. Several analogous systems are found that likewise feature a signature motif on the extracytoplasmic face, a C-terminal transmembrane domain, and cluster of basic residues on the cytosolic face at the protein's extreme C-terminus. The PEP-CTERM/exosortase system, found in many Gram-negative bacteria, seems to be related to extracellular polymeric substance production. The PGF-CTERM/archaeosortase A system in archaea is related to S-layer production. The GlyGly-CTERM/rhombosortase system, found in the Shewanella, Vibrio, and a few other genera, seems involved in the release of proteases, nucleases, and other enzymes.


Mitochondria, 3rd Edition

Pin-Chao Liao , . Francesco Pallotti , in Methods in Cell Biology , 2020

1.1 Definition

Mitochondria from even a single region of the brain are highly heterogeneous based on their morphological, histochemical, and enzymatic characteristics. Most methods are designed to isolate three distinct populations of mitochondria from rat brain: (a) non-synaptic mitochondria, the so-called “free mitochondria” (FM) (b) synaptosomal mitochondria (synaptic), which can be further subdivided into two fractions based on sedimentation properties, heavy (HM) and light (LM) ( Reijnierse, Veldstra, & Van den Berg, 1975 Van den Berg, 1973 ). Synaptosomal mitochondria are involved in regulating neurotransmitter release and synaptic vesicle formation. In contrast, non-synaptic mitochondria derive from multiple cell types and from neuronal soma and are involved in microRNA regulation and energy production ( Ly & Verstreken, 2006 Vos, Lauwers, & Verstreken, 2010 Wang, Sullivan, & Springer, 2017 ).


Open questions

Even though mitochondria and their membrane protein complexes have been studied intensely for more than five decades, they remain a constant source of fascinating and unexpected new insights. Open questions abound, many of them of a fundamental nature and of direct relevance to human health [61].

Concerning macromolecular structure and function, we do not yet understand the precise role of the highly conserved feature of ATP synthase dimers and dimer rows in the cristae and the interplay between the MICOS complex and the dimer rows in cristae formation. Are there other factors involved in determining crista size and shape?

We still do not know how complex I works, especially how electron transfer is coupled to proton translocation. What is the role of respiratory chain supercomplexes? Do they help to prevent oxidative damage to mitochondria, and if so, how? And how does this affect ageing and senescence?

We also do not know how the TIM and TOM protein translocases work, and what they look like at high resolution. The same is true for the structure of the MICOS complex at the crista junctions. How does it anchor the cristae to the outer membrane, and how does it separate the cristae form the contiguous boundary membrane? Similarly, the mechanisms of mitochondrial fission and fusion and the precise involvement and coordination of the various protein complexes in this intricate process is a fascinating area of discovery.

The biogenesis and assembly of large membrane protein complexes in mitochondria is largely unexplored. Where and exactly how do the respiratory chain complexes and the ATP synthase assemble? How is their assembly from mitochondrial and nuclear gene products coordinated? Does this involve feedback from the mitochondrion to the cytoplasm or the nucleus, and what is it?

And finally, how exactly are mitochondria implicated in ageing? Why do some cells and organisms live only for days, while others have lifespans of years or decades? Is this genetically programmed or simply a consequence of different levels of oxidative damage? How is this damage prevented or controlled, and how does it affect the function of mitochondrial complexes? Is the breakdown of ATP synthase dimers also an effect of oxidative damage, and is it a cause of ageing?

It will be challenging to find answers to these questions because many of the protein complexes involved are sparse, fragile and dynamic, and they do not lend themselves easily to well established methods, such as protein crystallography. Cryo-EM, which is currently undergoing rapid development in terms of high-resolution detail, will have a major impact but is limited to molecules above about 100 kDa [62]. Even better, more sensitive electron detectors than the ones that have precipitated the recent resolution revolution, in combination with innovative image processing software, will yield more structures at higher resolution. However, small, rare and dynamic complexes will remain difficult to deal with. New labeling strategies in combination with other biophysical and genetic techniques are needed. Cloneable labels for electron microscopy, equivalent to green fluorescent protein in fluorescence microscopy, would be a great help first steps in this direction look promising [26]. Once the structures and locations of the participating complexes have been determined, molecular dynamics simulations, which can analyze increasingly large systems, can help to understand their molecular mechanisms. Without any doubt, mitochondria and their membrane protein complexes will remain an attractive research area in biology for many years to come.


The number of mitochondria in cells can vary from a few pieces to thousands of units. Cells, which are making the synthesis of ATP molecules, have a greater number of mitochondria.

Mitochondria have different shapes and sizes, there are rounded, elongated, spiral and cupped representatives among them. How big are mitochondria? Usually, their shape is round and elongated, with a diameter from one micrometer to 10 micrometers long.

Mitochondria can move through the cell (they do this thanks to the cytoplasm) and remain motionless in place. They always move to places where energy production is needed the most.


Mitochondrial OXPHOS complex assembly lines

Mitochondria are critical for cellular energy generation and house oxidative phosphorylation (OXPHOS) complexes, which are under dual genetic control. A study finds that transcript translation and complex assembly are partitioned, and OXPHOS complexes III, IV and V are built at spatially defined regions of the mitochondrial inner membrane.

Mitochondria are essential for ATP production through oxidative phosphorylation (OXPHOS) 1 . OXPHOS complexes I–IV are located in the tubular membranes of mitochondrial cristae and complex V (F1F0–ATPase) localises to cristae bends 1 . However, it is unclear how OXPHOS complexes are spatially assembled. Using super-resolution microscopy, Stoldt et al. 2 now find that complexes III and IV commence assembly at mitochondrial inner membrane locations that are spatially distinct from their final destination. In contrast, complex V appears to assemble directly within cristae membranes. These findings provide insights into the spatial organization of membrane-protein-complex assembly within mitochondria.


Watch the video: Πόση πρωτεΐνη μπορούμε να απορροφήσουμε ανά γεύμα; (June 2022).