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0:08
My name is Tom Rapoport. I'm at the Department of Cell Biology at Harvard Medical School, and also a Howard Hughes Medical Institute Investigator. Today, I will be talking about basic principles of organelle biosynthesis. And I have a second lecture in which I will be talking about the morphology of organelles. Okay. So, let's start out by reminding you that every eukaryotic cell is a very complex thing. It contains a lot of membrane-bound organelles inside.
0:39
So, the most... the most prominent and largest organelle is the nucleus in the cell, which is surrounded by two membranes. It has actually pores through which material can get in and out. Inside the nucleus, of course, you have the DNA. The DNA is transcribed into mRNA. The mRNA is exported into the cytoplasm, and then translated by ribosomes into proteins. In addition to the nucleus, we have organelles called the mitochondria, which are the powerhouses in the cell.
1:10
So, they are surrounded by two membranes. They are generating most of the energy in the cell. And there are organelles called the lysosomes, which are degrading proteins and other material. Then there are peroxisomes, which are surrounded by a single membrane, and they are involved in lipid metabolism. They are also involved in detoxification, particularly of hydrogen peroxide. There's an organelle called the Golgi apparatus. It consists of membrane sheets that are stacked on top of each other.
1:43
And then finally, my beloved organelle is the endoplasmic reticulum membrane, which we also call... abbreviate by ER. This is a membrane system that extends throughout the entire cell. It consists of membrane tubules and membrane sheets. So, every organelle in the cell has a particular task for the overall well-being of the cell. Now, this achievement of having many organelles in the cell is actually something that didn't exist forever, and is today also not seen in more primitive cells,
2:16
like prokaryotes. So, in this case, you don't have any membranes inside the cell. So, in this case, you have just the plasma membrane, and there are no membrane-bound organelles inside. So, during evolution, the first thing that happened is that there were some membrane invaginations, and then eventually these membranes pinched off, and this generated the nucleus and the endoplasmic reticulum. And then later in evolution, these primordial cells took up an aerobic bacterium,
2:52
and that gave rise, eventually, to mitochondria. And then in plant cells, there was another endosymbiotic event. A cyanobacterium was taken up, and this gives rise, now, to chloroplasts in nowadays' plant cells. So, here is a very important point. Most organelles are actually generated from the same type of organelle. And this is similar to a statement that Rudolf Virchow made a long time ago. In Latin, it's called "omni cellula e cellula," which means "cells are only generated from cells."
3:34
This shows Rudolf Virchow. I have a particular attachment, actually, to Rudolf Virchow, because he did most of his work in Berlin, at the Charité, which is the major hospital in Berlin. And I myself grew up in Berlin, and actually did my PhD at the Charité. Rudolf Virchow was really a remarkable person. He was not only a scientist. He was also a physician. And he was a politician. Okay, well, Rudolf Virchow was not the first person to actually coin this famous sentence, "omni cellula e cellula."
4:08
In fact, this dates back more than 30 years. And the so-called cell theory, which means that cells can only be generated from cells, was actually formulated first by these two German scientists, Mathias Schleiden and Theodor Schwann. The story goes that they were good friends, and they met at a dinner place, and Schleiden said, you know, I'm looking at plant cells, and I see never anything that is alive without seeing cells. And then Schwann said, you know what?,
4:42
I see the same thing with animal cells. And so this was the birth of the so-called cell theory, in 1838. And basically, it took about 30 years for everybody to accept that idea. So, back to the organelles. So, as we said, cells can only be generated from cells. And the same is true for many organelles in the cells. For example, the nucleus can only be generated from the nucleus. Mitochondria can only be generated from mitochondria, chloroplasts only from chloroplasts,
5:17
and the endoplasmic reticulum only from endoplasmic reticulum. And the principle is always very simple. You start out with a small organelle, it grows by taking up proteins and lipids, and then finally it divides into daughter organelles, and you start over again. There is an important consequence of this very simple principle, namely that during cell division each daughter cell needs at least one copy of the organelle. Otherwise, you cannot propagate the organelle.
5:48
But there are different strategies for how the different organelles are propagated. So, in the case of the nucleus, there is a very precise coordination with cell division. So, during cell division, each nucleus divides, and you generate two nuclei. And so... simultaneously with a cell division. In the case of mitochondria, it's a completely different strategy. Here, you generate enough mitochondria in the mother cell such that during division -- cell division --
6:21
you have sufficient numbers in the daughter cells so that each daughter cell gets at least one copy. The idea is that you need about 300 copies of mitochondria to be sure that every daughter cell has a copy. And then in the ER case, it's still a different mechanism. In this case, during cell division the ER is simply torn apart, and each daughter cell gets some aspect of the reticular network. The strategy is not entirely clear for the Golgi apparatus.
6:54
In this case, there are two different ideas. In one case... one idea is that the Golgi fragments during mitosis, and then it reforms after mitosis from fragments. So, this would be very similar to what we just saw for mitochondria. The other possibility is that the Golgi is absorbed into the ER during mitosis, and then reforms from the ER after mitosis. And it's probably a combination of the two mechanisms that is actually really operating here. In the case of peroxisomes,
7:32
it's very clear that these organelles are not entirely autonomous, because the membrane proteins are first inserted into the ER and then moved to peroxisomes, whereas luminal proteins are imported directly from the cytosol into peroxisomes. So, in this case, the peroxisomes cannot be generated from peroxisomes alone. They have to rely on the ER. There's another consequence, namely that during zygote formation... the egg brings all the... all... basically all organelles,
8:05
because it brings most of the cytosol, most of the cytoplasm, whereas the sperm brings essentially only the nucleus. So, this is schematically shown here. So, during the zygote formation, essentially all the mitochondria and all the ER come from the mother. Now, unfortunately we cannot really see this for every organelle, but for mitochondria we can because they have a DNA, and so the DNA tells us that mitochondria come only from the mother. Now, each organelle in the cell
8:38
is characterized by a specific set of proteins, and that is quite obvious because each of these organelles has a specific function, and these functions are carried out by specific proteins. And as a general rule, every protein is only found at one location in the cell. Of course, when a protein is on its way to its final localization, then it might be in a different location, but generally speaking, and then finally, it's always in one location only. And most organelles have
9:09
both luminal proteins and membrane proteins. Membrane proteins sitting in the membrane, and luminal proteins being in the inside... soluble in the inside. The only exception is the Golgi apparatus. In the Golgi, we don't have soluble resident proteins. If you have soluble proteins in the Golgi, they are in transit through the Golgi to another destination. Now, lipids are also localized like proteins, but not as exclusively. There are some lipids that are known to
9:41
be very enriched in certain places. So, for example, cholesterol is high in the plasma membrane and low in the endoplasmic reticulum, where it's actually being made. Phosphoinositides are also found in different organelles. For example, phosphoinositol... the PI(3)P phosphoinositide is found specifically in endosomes. And other phosphoinositides are found in the plasma membrane or in lysosomes. And finally, cardiolipin is probably the most specifically localized lipid in the cell.
10:13
It's only found in mitochondria. So, now I will be talking about how proteins are transported in the cell. So, how do they get to their final destination? Now, if you think about it, then most proteins in the cell are actually synthesized in the cytoplasm on cytosolic ribosomes. But eventually these proteins need to be transported to many different sites in the cell, as we discussed, in chloroplasts, mitochondria, nucleus, and so on. So, there must be signals
10:50
that are required to direct proteins from the common site of their synthesis, in the cytosol, to their final destinations. And it's simply like a zip code system -- same zip code means same destination. So, with this concept, we don't need any signal sequence for proteins that actually stay in the cytoplasm, because they just are synthesized in the cytoplasm and they just stay there. In the case of nuclear... nuclear proteins, they need a nuclear localization signal
11:22
to direct them from the cytosol into the nucleus. In the case of mitochondria, we need mitochondrial signal sequences to direct them into mitochondria. Now, I mentioned that mitochondria actually have two membranes, so this generates subcompartments. There's a compartment in between the outer and inner membrane, and there's also the matrix space. And so a single signal is actually not sufficient in the case of mitochondria, because you need first a signal to direct them to mitochondria,
11:50
and then a second signal to direct them into a subcompartment of mitochondria. And then finally, we have this large class of proteins that need an ER signal sequence. So, these proteins are initially translocated across or integrated into the endoplasmic reticulum membrane. And then from there, they can be transported to other organelles in the cell. So, let me explain this in a little bit more detail. So, I'll be talking about protein translocation across the endoplasmic reticulum membrane.
12:25
So, as I mentioned, there is a large class of proteins, probably about 20% in every cell, that are transported, or translocated as we say, across the endoplasmic reticulum membrane. Some proteins make it all the way across the membrane and end up in the lumen of the endoplasmic reticulum, shown here in this red thing. And other proteins are integrated into the membrane and become membrane proteins. In a second phase, proteins are transported by vesicular transport
13:00
from the ER to other destinations. So, initially you have vesicle budding from the endoplasmic reticulum. These vesicles contain the soluble protein in the lumen and the membrane protein in the membrane. These vesicles then go to the Golgi apparatus. They fuse with the cis side of the Golgi, and deliver the soluble protein into the cis cisternae, and the membrane protein will then become a membrane protein of the cis cisternae. And then by subsequent...
13:30
successive vesicle budding and fusion events, the protein moves through the Golgi until, at the trans side of the Golgi, you again have vesicle budding. These vesicles then go to the plasma membrane. And then, when they fuse, they deliver the soluble protein to the outside of the cell -- we call this a secretory protein -- and the membrane proteins will become plasma membrane proteins. Now, of course you may need proteins that also are resident in the ER or the Golgi apparatus,
14:01
or they may be also transported to other organelles -- endosomes and lysosomes. And those proteins then need additional signals -- retention signals or sorting signals -- to go to these other destinations. So, now I will focus on the first step, the translocation step, and tell you a little bit more in detail how this works. So, just to remind you again, proteins can be either translated completely across the membrane and end up in the lumen, or they can be integrated into the plasma membrane.
14:37
Proteins that are translocated into the lumen have a signal sequence. And that signal sequence is... has as a major characteristic 7-12 hydrophobic amino acids in a row. In contrast, transmembrane proteins have transmembrane segments, which I will abbreviate by TM segments, and those segments are usually 20... about 20 hydrophobic amino acids in a row. But both types of proteins are integrated by... or translocated by the same mechanism, the same channel. I want to make the point here that
15:17
translocation in bacteria or in archaebacteria occurs by a very similar mechanism. This process is really extremely conserved. It's one of the most ancient mechanisms that exists. As soon as you generated a cell, you needed a translocation system. So, in bacteria, we distinguish gram-negative and gram-positive bacteria. Gram-negative means that they have two membranes surrounding the cytoplasm. And secretion in bacteria means that you have a signal sequence
15:48
and you direct the protein across the inner membrane into the membrane space... into the aqueous space in between the membranes, which is called the periplasm. In the case of gram-positive bacteria, you translocate through the plasma membrane and the protein ends up in the medium. But the important point here is that the signal sequences -- or in the case of a membrane protein, the transmembrane segments -- are essentially the same as in eukaryotic systems.
16:16
Now, it's not a surprise that it's similar in... that the transportation system is similar in bacteria and in eukaryotes. If you consider again how these... or, how the ER was generated during evolution, you started out with a primordial single-cell organism where translocation happened through the plasma membrane -- like in today's prokaryotic cells, bacteria -- and then you got an invagination, and then finally this membrane pinched off,
16:49
and now the translocation occurs through an internal membrane, the endoplasmic reticulum membrane. And so it's not a surprise that this translocation system is basically the same as the primordial translocation system in single-celled organisms. Now, how does this actually work? Now, a major step forward in this field came with the postulate of a signal hypothesis. This was work by Gunter Blobel and Bernhard Dobberstein, who published a very influential paper,
17:24
and this is a scheme that I took from that paper. So, you're starting out with the translation of secretory protein, here. The ribosome starts at the 5' end of the mRNA, at the AUG codon, and then moves towards the 3' end, synthesizing the N-terminus first. And this dotted thing at the... at the beginning of this line is supposed to mean that this is the signal sequence. So, the signal sequence directs the ribosome to the membrane. The ribosome binds to a channel
18:00
that is shown here with these yellow dots. And then the polypeptide chain is moved through the membrane as it is being made by the ribosome. Eventually, the single sequence is cleaved off, so you generate a new N-terminus that is now in the lumen. The protein will then be completed, and end up on the... in the lumen of the endoplasmic reticulum. The ribosome eventually dissociates into its two subunits, and then you can start a new cycle. So, this shows in a little bit more detail
18:32
how these signal sequences are structured. I mentioned already that the important part of a signal sequence is this H region, a segment of at least 7 but usually between 7 and 12 hydrophobic amino acids in a row. There's also an N region, which is positively charged, and a C region, which is also hydrophilic. And then there is a cleavage side, which is characterized by two small aliphatic regions... amino acids at positions -1 and -3. So, about 5 amino acids away from the hydrophobic region,
19:12
and then you have these two small aliphatic residues. Then, this signifies where the signal sequence will be cleaved by an enzyme called signal peptidase. So, the mechanism that I described we call co-translational translocation. What it means is that the ribosome sits on top of the channel -- here's the ribosome -- and that tunnel inside the ribosome, through which the polypeptide chain is moving, is aligned with a membrane channel, shown here in red, such that the polypeptide chain
19:44
has only one way to go. The channel is... we call it Sec61 channel in eukaryotes and SecY channel in prokaryotes, but essentially it's the same. So, the names are different, but the composition and the architecture of the channel is always the same. This channel is formed from a heterotrimeric membrane protein complex that contains a large alpha subunit and two small beta and gamma subunits. This shows the X-ray structure of the channel, determined for an archaebacterial channel, but, as I said,
20:24
it's essentially the same for the Sec61 channel in higher organisms. This was a major achievement, if I may say so, that we were able to do... it's almost now 14 or 15 years ago. So, what... on the left panel, this panel here, you're looking down onto the channel. You're sitting in the cytosol and looking down onto the membrane. And you can appreciate that the alpha subunit of the channel, the large one, consists of two halves, shown here in blue and red. Transmembrane segments 1-5 in blue,
21:01
and 6-10 in red. They're linked at the extracellular side by a loop between TM 5 and 6. The gamma subunit wraps around the two halves and keeps the two halves together. And we call this side, here, the front side, or the lateral gate. So, this channel can open up like my fingertips do. So, it's similar to a clamshell, that it can... opens up sideways. We call this the lateral gate. Now, an inside view... you can appreciate that the channel has an hourglass shape
21:35
with a cytoplasmic funnel that is empty, and an extracellular funnel that is filled with a little helix, shown here in yellow, that is... that we call the plug. And in the center of the membrane is a constriction that is formed by 6 hydrophobic amino acids that project their side chains radially inwards. And we call this the pore ring. So, this shows a scheme of how this channel actually is put to work. So, we're starting out with a closed state of the channel.
22:08
This is with the state number 1 on the slide. Then, as the next step, the ribosome binds to the channel. This somehow destabilizes the channel already. And then in the next step, step number 3, the nascent polypeptide chain is inserting into the channel as a loop, with N and C-termini staying in the cytosol, and the signal sequence located in the lateral gate. Then, the signal sequence stays put, whereas the C-terminal part of the hairpin is moving through the channel.
22:39
Eventually, you get signal sequence cleavage. The polypeptide chain is then completely in the lumen. The ribosome moves and eventually dissociates. I forgot to say here that during the... when this nascent chain inserts, the plug moves towards the back, and this allows the channel to be opened. So, this shows the actual structure in which the polypeptide chain is seen in the channel. This is the blue line that you see in the channel. This is a cut through the channel.
23:11
This is an X-ray structure that we obtained a couple of years ago. And you can appreciate that the polypeptide moves in an aqueous environment. Essentially, there's water everywhere, so the polypeptide chain is never moving, actually, through the hydrophobic interior of the membrane. And the hourglass shape of the channel minimizes contact of the polypeptide with the channel, such that you need very little energy to move the polypeptide chain through the hydrophobic membrane.
23:41
Otherwise, it would be very, very energetically costly. So, how is the signal sequence then recognized by the channel? And the principle of how it is recognized is actually quite simple. So, here you see a struct... the same structure, actually, but now we're looking at the signal sequence, shown here in color. In green is the H region, the hydrophobic region. And you can see that it's outside the lateral gate, facing the lipid in the membrane.
24:16
And so this principle of signal sequence recognition is very simple. It is simply partitioned into the lipid phase. It's hydrophobic enough so that it likes to be in a lipid environment. It's a very primitive recognition. All that happens is that the hydrophobic amino acids of the signal sequence like to be in the lipid environment. The N-region, the positively charged region that I mentioned, is located on the cytoplasmic side, and the C-region, with the cleavage site, is on the luminal side.
24:51
So, now I'm gonna talk about how membrane proteins are integrated. What I told you so far is how proteins move all the way across the membrane, in the case of a secretory protein. Now, let's look at a membrane protein integration. And the principle again is quite simple. So, here, on the left side, you have a protein moving through the aqueous interior, but when a hydrophobic segments arrives, shown here in yellow, it moves sideways out through the lateral gate into the lipid phase,
25:24
and becomes a transmembrane segment. And this will then generate both luminal and cytoplasmic segments of the transmembrane protein. at any given point the hydrophobicity of the polypeptide segments inside the channel is being probed. If the hydrophobicity is high enough, then the polypeptide chain will move out sideways into the lipid. If not, it will stay in the aqueous channel and move on to the luminal side of the ER. So, as a... and different from the secretory protein,
25:57
which all move all the way through the membrane, and so there's no topology problem. But in the case of membrane proteins, you can have many different types of topologies. And we distinguish four types of membrane proteins depending on their topology. Type I has the N-terminus in the lumen and C-terminus in the cytoplasm, and they also have a cleavable signal sequence. Type II and III have no cleavable signal sequences, but they have a transmembrane segment close to the N-terminus
26:28
that remains uncleaved. We also call the segment "signal-anchor sequence." And then Type IV just summarizes all multi-spanning membrane proteins, and obviously they can have many different topologies. They can have, you know, any number of transmembrane segments, from 2 to 12 or even more. And then the N-terminus can either in the... in the cytosol or in the lumen. So, let's have a look at how these proteins are actually synthesized. So, let's start with type I membrane proteins.
27:02
As I mentioned, they have a cleavable signal sequence, and then they have downstream a stop-transfer sequence. So, we're starting out with the ribosome synthesizing the polypeptide chain. The signal sequence emerges from the ribosome. And you get insertion of the protein as a loop into the channel. This is exactly the same as we saw it for the secretory proteins. At some point, you get signal sequence cleavage. And now we're assuming, in this case,
27:33
that there is a hydrophobic sequence following... at the C-terminal... at the C-terminus of the protein, shown here in yellow. And when that hydrophobic sequence arrives, it moves sideways out through the lateral gate into the lipid. And now the ribosome is synthesizing a cytosolic fragment... a cytosolic segment of the polypeptide chain. And then eventually it terminates translation. And so you have a C-terminus in the cytosol, the N-terminus... a newly synthesized...
28:02
the newly generated N-terminus after signal sequence cleavage is in the lumen. That's how type I membrane proteins are synthesized. I should say that some people call this C-terminal transmembrane segment also stop-transfer sequence. So, the next class is type II membrane proteins. So, they start exactly the same way. So, the hydrophobic sequence emerges from the ribosome. Now, the difference is that this hydrophobic sequence is a little longer than a signal sequence
28:36
-- remember, 20 hydrophobic amino acids rather than maximum 12. And also, in the class II proteins you have positive charges preceding the hydrophobic transmembrane segments. So, this polypeptide chain inserts as a loop into the channel, similar as with the secretory proteins. But the difference is now that the hydrophobic sequence moves all the way out into... laterally into the lipid phase, the N-terminus stays in the cytosol, the ribosome synthesizes the protein,
29:07
and it's moving into the lumen of the ER. And then finally, after termination you have the C-terminus on the luminal side and the N-terminus on the cytoplasmic side. These are the type II membrane proteins. Type III proteins are similar, but in this case, the positive charges are actually succeeding the hydrophobic sequence rather than proceeding it. And what happens is that now the N-terminus of the protein flips across the membrane right away,
29:41
and the hydrophobic sequence moves out of the channel into the lipid phase right away. And now the ribosome is synthesizing the cytoplasmic segment of this polypeptide chain. So, eventually you have a polypeptide chain where the C-terminus is in the cytoplasm and the N-terminus is in the lumen, so, the exact opposite orientation as the type II membrane proteins. Now, in order for this flipping to happen, the N-terminus cannot be folded and cannot be very long.
30:12
So, there are some restrictions to this mode of... to this class of membrane proteins. And then finally we have type IV membrane proteins. And as I mentioned, there are many of them, and I've just picked an example where you have 3 transmembrane segments and the N-terminus stays in the cytosol. So, let's go through this here. So, we have the hydrophobic segment emerging from the ribosome. Positive charges in this case are preceding. So, you get a loop insertion.
30:43
The positive charges keep the N-terminus on the cytoplasmic side. So, the ribosome is now moving the polypeptide chain into the lumen of the ER. At some point, you make the second transmembrane segment. It moves then, also, sideways out, shown in the lower panel. Then the next hydrophobic section emerges. You get, again, loop insertion of the polypeptide chain. And then, if there's no further transmembrane segment being made, the C-terminus ends up on the luminal side,
31:14
and you get a triple-spanning membrane protein with N-terminus in the cytosol and C-terminus on the luminal side. Now, this mechanism implies that there is a sequential insertion of membrane prot... of the transmembrane segments. This is the simplest model, I have to say. However, you would expect, for example, that if you take out the transmembrane segment of a protein that everything downstream would invert its topology. And this is in fact the case in some proteins, but not always the case.
31:45
So there's... it's not always as simple as I depicted here in these schemes. So, as the very final point in this presentation, I want to bring up this very interesting and very important point. How do you maintain the membrane barrier for small molecules when you're translocating a large protein? If you think about it, it's not a trivial thing to maintain a barrier for small molecules while you're transporting a large thing. And you need to do that, because in the case of the ER
32:18
you have a high calcium concentrat... calcium ion concentration in the luminal ER, and you don't want the calcium to go out into the cytosol while you're transporting a protein. Now, in bacteria it's even more serious, because you have a membrane potential across the inner membrane, which is the major energy source for the cell. So, how do you maintain that barrier? And this... the mechanism is very simple. It was worked out by a former student in the lab, Eunyong Park.
32:46
So, you're starting out with the closed state of the channel. And this channel is closed both by the pore ring and by the plug sitting underneath the pore ring. So, no flu... ion can go through because of these features. Once you have a translocating polypeptide chain in the channel, the plug is out of the way. It moves towards the back, as I mentioned before. But now the pore ring residues form a gasket-like seal around the polypeptide chain.
33:16
And that prevents the free flow of ions through the membrane. Eventually, the polypeptide chain leaves the channel, either towards the other side of the membrane, as shown here, or in the case of a membrane protein, you can move it sideways out into the lipid phase. And in both cases, now the plug has a chance to go back to the center of the channel and reseal the channel. So, under no circumstances is the channel ever conducting ions and small molecules.
33:47
So, this brings me to the end of this presentation. I just want to make sure that we go back and understand what we tried to cover here. So, as I mentioned, we have in the eukaryotic cell many different organelles. Each of these organelles has a certain set of proteins. They're all... most of them, at least, are synthesized in the cytosol, and then have to be transported to different sites in the cell. And we were mostly concentrating on the major export pathway in the cell,
34:18
the endoplasmic reticulum, which then gives rise to the proteins sitting in many other organelles. So, it's an interesting system. I will have another lecture coming up, which will deal with how these different organelles actually generate their different morphologies, which is an equally interesting question.
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