 recipient in 1999 of the Nobel Prize in Physiology or Medicine and also John D. Rockefeller Jr. Professor at Rockefeller University in New York City. The Nobel Committee recognized Dr. Blobel for the insights that he and his students accumulated over a couple of decades that told us about the built-in information that directs a protein molecule to its intended destination in the cell. Each protein has an address label either at one end his original idea almost just about 30 years ago or found among the many twisting bumps and crevices on a protein surface. This kind of molecular zip code helps the protein wend its way through the tunnels that move through the cells interior. The protein may need to insert into a membrane as Dr. Fisher has shown us or join other molecules in the power-generating mitochondria or the light-capturing chloroplast. Despite the pashaing of other cell biologists, Dr. Blobel found channels constructed by protein molecules that other proteins use as a conduit to move through the oily interior of internal membranes. He has also studied the protein-lined pores that perforate the two membranes that in turn enclose the nuclear material channels through which one million molecules pass each second. There's kind of an irony in this kind of an irony in here when Dr. Blobel arrived at the Twin City Airport, his luggage didn't arrive at the correct destination despite the barcode label that was intended to direct the luggage to the appropriate carousel. He was born in a small forming town in Silesia, Germany, the son of a veterinarian. As World War II drew to a close, the advances of the Russian army forced the family to flee further west into Germany. On the trek, Dr. Blobel and his family watched as Allied bombs leveled Dresden, that once great city of splendorous architecture. As a young man in what was then East Germany, Dr. Blobel changed his address to West Germany so that he could continue his studies. He said that he was able to do that before the Berlin Wall had been put up to keep people like him from moving. After completing a medical degree at the University of Tümmingen and a stint as a medical intern, he felt restrained as a medical practitioner because he was handling only symptoms and not the molecular reasons for a disease. So he attempted to find funding to support graduate degree in Europe, but was not successful either, even unsuccessful in the Fulbright, but had a brother, has a brother who at that time was on the faculty at the University of Wisconsin, and so his brother encouraged him to go to the University of Wisconsin. While Europe's loss, the United States gained. At the University of Wisconsin, he completed a PhD in the study of cancer oncology and then moved to Rockefeller University where he worked in the lab as a postdoc in the lab of Dr. George Pilata, who was later to become also a Nobel laureate, sort of as we heard from Professor Norby, Nobel laureates beget Nobel laureates. And it was in this time that after, as a result, probably of Dr. Pilata's interest in the traffic of proteins that Dr. Global figured out how proteins know where to go. He's a very generous person. He donated some of the money that accompanies the Nobel Prize to an international foundation that is raising $200 million to reconstruct the Frau in Kierke, the Protestant Church of Our Lady in Dresden, which you'll see up here on the two slides, the original Frau in Kierke. He also donated money, some of this money from the Nobel Prize for rebuilding a Dresden synagogue that had been burned by the Nazis in 1939. The prize money that came from the King Faisal Award, another award in his, the awards that Dr. Global has received also went to the Dresden Foundation. The money that he's donating is going to the American wing of that reconstructed cathedral. And at breakfast this morning, I hope you don't mind me saying this, but at breakfast this morning, Dr. Global told us that the honorarium for his contributions to this conference will be given to reconstruction of a seventh-century mosque in Yemen. And so we look forward to, we look forward to hearing about your studies on these, these informational molecules that the cell uses for destinations and what lies ahead. Dr. Global. Thank you. I would like to thank the organizers of this meeting, particular Professor Stoyer for inviting me. And it's one of the great meetings I have attended. The organization is just flawless and it has been two wonderful days. And I would like to thank everybody here from, from the little choruses last night and the noble symphony to, to slide projections. It has always been very perfect. Now, don't worry. I'm not going to give a talk about Dresden. Also, I like to talk about it. I just wanted to point out that this is probably the Fraunkeche is one of the most imposing buildings north of the Alps and by many considered one of the most important. And what you see here is not just a half sphere as a cupola, but it's a bell. The, the, the entire church is in form of a stone bell, which is about 100 meters high and therefore dominated the Dresden skyline and was of course painted by numerous painters. And it's really the, the determining feature of the Dresden skyline and it's up to here. It's now built up to this point. It's the inside is round and it's very high and it has five tiers. It's almost like an opera house and it has of course a very good acoustic and we are right here now. And in four years, we will be finished. There's still a 60 million, 30 million dollars missing. So if anybody of you has a lot of money, get in, get in touch with me and something will be built that will hopefully last for a long time to come. So we have heard a lot about proteins today and the previous speakers have introduced the subject. We have heard about one, one protein that is the preon protein that Stanley Prusina described and we have seen the complications enganded by one single protein and how little we know about this one little protein. And now we have many, many Edwin Fischer mentioned maybe a million different proteins in the cell. So we are facing a tremendous challenge. The term proteomics has been coined as we discussed. I think, you know, the next term will be cellomics. Cellomics, you know, the various cells that we find in the living universe. As you know, the cell rose from 3.5 billion years ago. It took about 1.5 billion years. Earth is 5 billion years old. It took about 1.5 billion years to develop a cell. Nobody knows how the cell came about. But what is very interesting is that the cell has divided ever since. Of course, it has changed dramatically. But certain features of the cell that were, that arose 3.5 billion years ago are still present. For instance, the membrane which surrounds the cell is probably very similar to then the membrane that we had 3.5 billion years ago. It's a bilayer of lipid molecules and I will come back to that. So the really wonderful thing is that these cells have divided ever since. And you of course are a product of this cell division and cell association. So from a cellular point of view, you are 3.5 billion years old of continuous cell division. Think about it. Your mother, your grandfather, you came from a cell and at some point these cells lived in animals and they live in plants and they were bacteria. And this is this wonderful relationship between all life. And this is not, this inside is not very old. It's about 100 years old. When a German pathologist Wilchow postulated in the 1870s that omnicellular acellular, he said it in Latin, each cell comes from a pre-existing cell. Before people thought that cells can come from some secreted substances and you can form extracellular, you can put together from secreted substances a cell. And we now know that this of course is not possible. And so that we all have adopted omnicellular acellular, each cell comes from a pre-existing cell. And so as you are sitting here, you are from a cellular point of view 3.5 billion years old. Now let me tell you a bit more about cells in general before I go into my own this specific topic. And I wanted to show you here an axel. And you can see this huge axel. And it is being, there's a sperm has come in with its long tail. You can see the long tail here. And this is a sperm head where the genetic material is. You can see how insignificant this sperm looks by comparison to the axel. As you know, the mitochondria, which give you the power, the ATP, and we have heard about ATP, come from the mother. So you inherit the mitochondria from the mother. There is literally no mitochondria, which add in the fertilization. And this is how you start out life. You start out life as a single cell. And from then on, you undergo a number of divisions, about 10 divisions or so, even less. And you form a little blastocyst, which is not bigger than this egg cell. And the blastocyst is a little hole, which is surrounded by cells. And it is a little vesicle, I would say, surrounded by cells. And there is a little cell mass in there. I should actually have a picture of that. And that are the so-called embryonic stem cells. And we will talk a bit later about embryonic stem cells. Of course, these are very important cells, because they are totally potent. These cells can develop into any cells in our body. But they cannot form another embryo, because the cells that are required for that are part of this vesicle. So you see this. And in the next slide, you see this a little bit more close up. You see these many villi here. And you see the tail of the sperm here. And you see how the sperm head has, the membrane of the sperm head has fused with the plasma membrane of the egg cell. And then what happens is there are only about 50 divisions necessary to form a human body. Only 50, which you calculate. It's several hundred trillion cells in our body. And these cells then specialize. And here is a bunch of blood cells. And you see the red cells here, which look like pennies. And then the white cells, which look completely different. They have these ruffles on the surface. And here you see something which is very striking. You see a normal red cell, and you see a sickle cell. And all that happened in the sickle cell is that the globin molecule, which is a protein in these red blood cells, has one amino acid changed out of 170 or so, that is changed into another amino acid. And this one single mutation causes the cell to change shape. The globin actually is not losing its capacity to do its work. But it changes the shape of the cell into this sickle-like shape. And these sickle-shaped cells cannot navigate through the very narrow capillaries in the brains elsewhere, elsewhere in the body. And therefore they are mechanically prone to destruction. And so these people who have this disease, sickle cell anemia, have an anemia. They cannot make as many red cells as are being destroyed constantly. They just make a bit more than are being destroyed constantly. Of course, this mutation was a very useful one once in this globin molecule because it protected the people who had this mutation from infection by the malaria agent. The malaria is a single cell and it has to attach to the red cell and has to be invaginate to enter the cell. And these sickle cells are much less effective in taking up the malaria, this malaria cell, in the plasmodium as it is called. And therefore these people who had these mutations survived better in the population because they weren't prone to malaria infection. So this is just a little story to telling you what, how important a one single protein can be and what advantages you may have from a mutation and what disadvantages you may have from a mutation. Now here you have cancer cells and on the surface they don't look that much different. If you look at cancer cells, they just grow on top of each other and they don't look that much different. And here you have a neuron with its many dendrites. And there are each of these dendrites you can see little buttons and each of these little buttons connect to another nerve cell. So each of these nerve cells may have 10,000 connections to other nerve cells. And there are more synapses, it is said, in the nervous system than there are stars in the universe. So it tells you something about the complexity of the human brain. Here is another way of showing these cells, these nerve cells with these many little dendrites and the many little butons as they are called, buttons which connect to other cells. And here is on the head of a needle is a bacterium. You can see the needle a little bit enlarged, this is just the needle a little pin that you would have in your suit and you can see the bacteria here and you can see them here more enlarged and here you can see them very much enlarged. And again they are very similar to two cells, to our cells, they have a membrane surrounding the cell and they have of course some other features which I won't go into. But the bacterial cells were the first cells that were made, that arose 3.5 billion years ago and this so-called archaebacteria and it is from there that these cells then have developed and have gone into becoming eukaryotic cells. So this is just giving you an idea in what environment proteins operate, namely in the framework of the cell. And now each time I go I'm going to go a bit more, the beautiful pictures will end and it's going to be a bit more challenging. So I will probably lose at each step as I go along 10 percent of my audience and then at the end I will show you some unpublished data and I will only probably keep two or three percent of the audience but I thought it would be nice to have something for everyone. So this is the easy part. Now you have already heard today about membranes and membranes were briefly mentioned but I think it's very important that you understand that membranes are made of lipid molecules and lipid molecules have oily tails and they have a water-loving head, a hydrophilic head and cholesterol, the dreadful cholesterol which everybody dreads is also one of these lipids and it has a similar, it has also a water-loving head and then a slightly different hydrophobic tail and these two get together and form a bilayer, a lipid bilayer and this is the fundamental organization of all membranes. You can see here the lipid molecule there is the water-loving head's face on one side and the water-loving head on the other side and in the center of this bilayer you have these hydrophobic water repelling tails of these lipid molecules and so nothing could potentially go through these lipid bilayers. They are a magnificent seal that nature has invented to seal the cell off from the environment but if you couldn't communicate with the environment it wouldn't be any good and so what has been done is proteins have been weaved in to these lipid bilayers and they span the bilayers and they form channels for instance for irons or even for water. Water doesn't get across spontaneously it needs a channel or it gets across very slowly but to do it faster you need a channel and then of course we have heard from Edmund Fischer about the many receptors for instance the insulin receptor and what is very important is that these membrane proteins are all a given membrane protein let's suppose a receptor that Edmund Fischer discussed today always has the same orientation in the membrane there has to be complete a symmetry of a given membrane protein and one of the big questions was how do you achieve this absolute symmetry for a given membrane protein and I will tell you what the solution is a bit later on. Now you can see when electron microscopy was really used to look itself and this happened at about the same time when Dresden was destroyed in February 1945 the first paper was published by Keith Porter, Albert Claude and Ernest Fulham from the Rockefeller University and they used the electron microscope to look at cells and what the cells look like. For hundreds of years people have stared through the light microscope and didn't see much than a membrane and the nucleus and if they used certain stains they saw several other spots in the cell but they couldn't really see and what the electron microscope showed for the first time that they are here the nucleus which people had seen in the light microscope is big enough to see it in the center but you see many many many membrane compartments the cell is chuck full of membranes so you could there's some cells which are 50 or 60 percent of the proteins will be membrane proteins proteins that are woven into the membrane and so these these tremendous number of membranes they signify various compartments of the cell so there are mitochondria I will show you in the next slide a bit more schematic here you have the nucleus and the nucleus has pores in their double endolob membrane and these pores are important for traffic between the cytoplasm and the nucleus and I will talk a little bit towards the end of my talk about this traffic there's about one million proteins and RNA molecules which per minute go in and out of the nucleus so there's a tremendous traffic going on and this traffic is very very important for another important finding that that I will come back to later on but here in pink you see these pink structures these are the mitochondria and they have numerous membranes and they're on these membranes ATP the universal currency of energy is produced in large quantities and so these these are the mitochondria there are many other organelles that I won't go and won't have time to describe you but what you can see that the cell is really truck full of membranes now in the nucleus you have the chromosomes you have the DNA that you all are familiar with and that you have heard of now here what I'm showing you is a single chromosome it's a human chromosome and we have in ourselves 23 pairs of chromosomes and you take a piece of DNA each chromosome has one molecule of DNA and it is very compact if you take the human DNA and and and look at the thin fiber it is two meters long and if you take all all DNA from ourselves you go back and forth to the Sun 300 times so this is how thin this thread is and the art now is to compact this thread in such a way that you can still make it accessible to machines which copy part of this thread and which to modify the DNA and here is the most condensed form of the DNA in the chromatin and this is done when you divide the cell and you want to distribute the chromosomes into daughter cells you need to have them very compact otherwise you would entangle the DNA and you you could never untangle it again so you have to compact it then when cell division is over you you you expand the DNA again and you expand only specific portion depending on what cell type it is you want to only play certain portion in the liver cell you play another portion of the DNA then you play in a in a in a pancreas cell or whatever so so each the portions then expand and here what has been done is an agent has been added to this chromosome and you now can see that the proteins the DNA is rolled around a protein complex the proteins have been removed and you now can see that the DNA here you can see still still see the chromosome you can see this skeletal network of the chromosome and you can see that the DNA has now spilled out in this tremendous long thread and here i'm showing you a magnification of this and you can see this thread of the DNA this is now without proteins it's just naked DNA and this DNA of course as you have heard in all red consists of four letters so the so the language is a four letter language and so the the sequence of these letters in these very long molecules has now been established we know this now okay now here i'm showing you some images of how the DNA is transcribed in messenger RNA molecules which leave the nucleus and get out of the nucleus and you see these beautiful structures where the transcription starts here at one end for instance you see very small molecules of RNA they become longer and longer and longer so they are the so-called Christmas tree structures so you can see that the molar the enzyme molecules when transcribed the RNA into messenger RNA and that's a very nice visualization so the DNA then is transcribed to these messenger RNA molecules or two other RNA molecules and then it leaves the nucleus to go to the cytoplasm and there it is translated by one of the most complex machineries that nature has in has has invented and that is the ribosome ribosome and i will show you some images the x-ray structure of bacterial ribosomes has just been published and i will show you some images later on on the on eukaryotic ribosomes but you then translate a four letter language into a 20 letter language and there is really a three letter code that goes into a one of the letters of the 20 of the 20 letter alphabet and the proteins are usually in the order of 400 or 500 of these of these amino acids long but there are some which much longer and some of them which are much shorter our words in english on the average are about seven letters so the proteins have many more letters and therefore can do many many more things and you have already heard about what they can do in during the day's lecture now here is george pallati was already mentioned before and he started to he was one of the pioneers in electron microscopy and he discovered many structures in the south and he worked out what is known as the secretory pathway and i'm showing you here the secretory pathway and these are it is the so-called endoplasmic reticulum this is one of the membranes that was discovered in 1945 when keys porter looked at the cell and he called these endoplasmic reticulum membranes they looked like a reticulum and so the name stuck and what was then found that these endoplasmic reticulum membranes have ribosomes attached to them and you already know that the ribosomes translate the messenger RNA and make proteins out of it and what pallata had found is and for this work he got the Nobel Prize is that proteins like insulin or growth hormone secretory proteins are made on ribosomes that are membrane bound and then he has traced back the pathway of these proteins from the endoplasmic reticulum all the way out of the cell and this pathway is called the pallata pathway or the secretory pathway so what happens is that the proteins while they're synthesized while they're translated by the messenger RNA on these ribosomes and i'm showing you the messenger RNA they are getting somehow across the membrane it wasn't known how and then they are packaged up in little vesicles little containers and these vesicles fuse with downstream compartments and then in downstream compartment sit many enzymes and the enzymes modify the protein they put on sugar molecules and they do all sorts of other things so it's very much like in the car assembly plan you have made just the carossary and as you go through these various compartments you put on other things to really make the fold the propane properly you make all sorts of other bonds to it and we have heard some of some of the enzymes already were discussed this morning also they are not in this compartment really and then eventually a vesicle takes a quantum of secretory proteins infuses with a plasma membrane and this is how the secretory protein gets out and so when i started in pallata slab as a post doctor fellow i was interested in the rather mundane question why do messenger RNAs for secretory proteins know that they have to be translated on these membrane bound ribosomes because there are many ribosomes in the cell free and why do they go to these membrane bound ribosomes a very fairly straight a trivial question and well there were many hypothesis i won't take you back into into history but an idea that we came up with was david sabbatini and here's this image in 1970 was a very simple one but even so there was absolutely no evidence for it and the idea that we proposed is that all nascent polypeptide chains that are made here's a messenger RNA is one end to five prime end of the messenger RNA and here's the three prime end of the messenger RNA and what happens at the first is small ribosomal subunit binds and then the large ribosomal subunit binds and then you have translation from from the from the messenger RNA into the polypeptide chain and what we postulated is that the polypeptide chain at the end has the X has a couple of amino acids which are unique to all secretory proteins and that sequence is then recognized by a binding factor which binds the translating ribosome to the membrane which is indicated here in this arch and then somehow it gets across the membrane we didn't speculate any further at this point was already enough speculation and then the proteins get somehow across the membrane then the ribosome comes off the membrane, recycles goes into a subunit pool and then the entire process starts again from the beginning and so so this is what we call signal hypothesis because what we said there is the information is in the messenger RNA but is translated into a sequence of amino acid at the amino terminus of the protein and that then recruits binding factors and other things in the membrane that we didn't spell out that then gets the protein across the membrane a bit later we added something to the to this whole idea namely that there is a channel a protein conducting channel in the membrane that consists of proteins itself and what we postulated is that the signal sequence acts like a ligand to open the channel and the ribosome helps in it to the attachment sites on the ribosome to this proteins to these proteins we have indicated here a trimer this was in 1975 of membrane proteins which are recruited from the membrane to form this aqueous channel so that that the protein can go across the reason why we did this very complicated scheme is because we wanted to keep the membrane in solute impermeable for other things because it turns out that on the other side of the endoplasmic reticulum membrane there are very high concentrations of calcium for instance and you don't want to have the calcium to leak out into the cytoplasm it would be a disaster and so we wanted to have this channel only to conduct nascent chains nascent polypeptide chains nascent unfolded proteins across so we attached a little signal sequence at the amino terminus which acts a key together with the ribosome to open the channel sounded all wonderful but it was sheer fantasy and of course all fantasies undergo severe criticisms which is only right and it took us quite a while to to actually come up with evidence for this scheme now the first bit of evidence which we obtained was together with a postdoctoral fellow with Bernhard Dorberstein we were able to set up a cell free system as it is called but you take the cell you grind it down and you isolate various components and you try to put it back together this is an old strategy that is being done in cell biology research and so what we did essentially we isolated nascent chain for a secretory protein we took some ribosomes and we isolated some membranes and we were in fact able after trying for two years we were able to reconstitute the whole thing so that in the cell free system in a little test tube this process this very complicated process could be repeated now once you're able to do that then you can analyze it you were hearing from from Stanley today that one person was asking the question can you reconstitute in the cell free system the conversion of the prion protein into an insoluble prion protein can you do it with isolate protein in the test tube and you can't do it that yet in this case it would be wonderful if we could do it because then we can learn what is going on you were able to do it in this case and this then led over a period of 15 years to tremendous new insights into tremendous new discoveries which I will go through with you now this is Peter Walter who made the first discovery and I asked him to send me a picture and he was obviously stimulated by Einstein also when Harry Croto showed the young Einstein picture there wasn't any tongue hanging out but you you remember the famous Einstein picture and so Peter was able to isolate what we had what we had postulated before namely that there is a binding factor which recognizes the signal sequence and it turned out that this binding factor was unexpectedly complicated it consisted of an RNA molecule and six proteins which we are binding to this RNA molecule and this we call the signal recognition particle because it functions as we had postulated in recognizing the signal sequence on the translating ribosome and to mediate the attachment of the translating ribosome to the membrane but we know that it attaches only to the endoplasmic reticulum membrane not to the mitochondrial membrane not to the Golgi membrane not to the plasma membrane so there had to be in the endoplasmic reticulum membrane a receptor which recognize the SRP so we postulate the existence of an SRP receptor and just before I show you how we isolated this I just show you that the SRP which you see here binds in fact to the signal sequence as it emerges this nascent chain traverses the large ribosomal subunit and the signal sequence is then recognized by one of the proteins which turns out to be a G protein of the signal recognition particle so this complex must then bind specifically to the endoplasmic reticulum membrane and there has to be a receptor and this receptor was isolated by Reid Gilmore together with Peter Walter when he was a postdoc in our lab and the SRP receptor turns out to be a dimer heterodimer also two G proteins so there are three G proteins involved in recognition of the signal sequence one in the SRP and in targeting it to the endoplasmic reticulum membrane it turns out this this SRP receptor is exclusively localized in the endoplasmic reticulum and in no other membrane so this very complicated process of signal sequence recognition and targeting by these components it gets the chain the secretory protein to the endoplasmic reticulum so this we understood at this point and here's just the summary so we have now bound the translating ribosome with SRP receptor in the membrane now the next experiments where is there a channel there were many other models you could imagine that there were models like the hydrophobic signal sequence partitions into the bilayer and then the free energy which you get from that is enough to get the rest of the chain across and you don't need a protein conducting channel and certainly Simon by electrophysiology and I won't show you the experiment he done they are very very beautiful but very complicated then found the fact found that there is a protein conducting channel and other work in other laboratories particularly in Randy Schachman's lab then isolated such a protein conducting channel and we now know what it looks like he has indicated the protein conducting channel it's called in the language of yeast sac 61 and so what then happens is when the the through a number of gtpa's events the signal sequence the SRP comes off from the signal sequence the signal sequence is now free to interact directly with the channel and it opens the channel and the ribosome attaches to the channel and now the chain can go across an aqueous environment now there are some other enzymes which are recruited to this channel ensemble which work while the chain is going across the membrane there is an enzyme called signal peptidase here in blue which cleaves off the signal sequence because you don't need it anymore and there are other many other enzymes I indicated just one of them which is recruited to this entire complex which puts sugar molecules on this nascent chain as it goes across now Emily Evans was a graduate student to purify the signal peptidase which was a very obvious work and very difficult to do and she succeeded I just wanted to show that we also were able eventually to isolate some of the other components and what I wanted to show you here is if I can is a cartoon now it disappeared from my screen we have to wait what happens which summarizes this whole thing the whole 25 years in an animated cartoon which lasts about three minutes it's very humbling if you can do that what yeah yeah yeah remember you just come home it's no longer on my screen right here okay all right so so I do it here okay now you have this small ribosome subunit you have a tRNA which brings in the first amino acid attached in the messenger RNA they bind first and they form this complex this is work that others have done that we have not contributed to then you get the large subunit to join the large ribosome subunit it's here in its joints and it it binds to the small ribosome subunit then more tRNA molecules come and this chain then has to test the worst the large ribosome subunit and you form you rotate the ribosome now you see the small ribosome and subunit in yellow the large ribosome subunit here and you see now a ballet of tRNA which delivers each one an amino acid so that the chain grows by one amino acid at a time and the colors indicate that each tRNA carries another amino acid and eventually you will see down here after this ballet is finished the one who did the animation like this tRNA ballet so it goes on for a little bit too long but in any case you can see that eventually there's energy utilized you see a little flash here gtp is hydrolyzed you see a little flash and eventually the chain will come out of the large ribosome subunit there is a tunnel in the large ribosome subunit through which the chain grows and here the signal sequence has come out and it's now available to bind sorry now i screwed up i have to go back and now advances very fast because i don't want you to take you to the same uh sequence again um go forward here and don't go or go on fast um here we have seen this already we have seen that part we have seen the tRNA ballet we have seen the chain coming out and now comes the signal recognition particle up here which joins and recognizes the signal sequence down here in binds and then comes this whole ensemble is then bound to the srp receptor and the srp receptor collides with the channel and then what happens then is that the channel is opened by the signal sequence and the ribosome attaches to it channel is opened and then um you you um start continue the translation and the chain is growing and signal peptidase has come and has cleaved off the signal sequence oligosaharital transferase which puts on the sugars puts on the sugar the chain gets across and finally it is completely across the memory and the channel closes the ribosome comes off now this took us 25 years you may you may say why did it take so long but anyway what what the interesting thing was that there are many other compartments in the cell which use essentially identical mechanisms they have a signal sequence of course it's different it is a different zip code because you want to go to a different compartment and they have signal recognition factors and they have channels and all of these where subsequently discovered in other systems either in our lab or in other laboratories so this gives you a a good idea um and now let's go on there is a very important thing that visual in gupah a graduate student discovered and that i mentioned already in the beginning namely membrane proteins how does the chain weave itself into the membrane and the idea was simply that they fold itself into the lipid bilayer they have somehow the information you make the chain in the cytoplasm and then the polypeptide chain folds itself into the membrane and this didn't sound reasonable so what we postulated and this is what visual in gupah has proven and i think this is a very important finding is that um the membrane proteins have a signal sequence undistinguishable from secretory proteins have a signal which addresses them to the membrane of the arm not showing all the components anymore and then what happens the channel is opened and part of the protein goes across to the other side but integral membrane proteins have an additional element sequence element which we have called the stop transfer sequence which is able to open this protein conducting channel laterally to the lipid bilayer so that this little segment the transmembrane segment can come out through this opening and get stuck in the bilayer and the rest of the chain is then remaining in the cytoplasm and if you saw Edmond Fischer's talk this morning and you have seen all the receptors there's always a portion that is on the outside of the cell which corresponds to this portion that has been translocated and there's a portion that spans the membrane and this is this portion and there's a portion that remains in the cytoplasm and it is by this sequence information in these membrane proteins that you stitch the membrane proteins into the membrane exactly for a given protein species as as the protein really dictates it to this machinery and the machinery just interprets this information in the nascent chain and this is unique this protein conducting channel because it can open across the membrane and it can also open in the second dimension to the lipid bilayer now we'll show you some low resolution images later on now here we come to Roland Beckmann and seeing is believing and we wanted to see this channel what does this channel look like and in the course of doing so he worked together with Christian Spahn from from Joachim Franks laboratory in Albany and they did some very beautiful work that is going to be published in a month from now in cell and this is the part that I will probably lose most of the audience but the five percent of the audience which can follow this will probably enjoy it what they did is they did a technique that has been invented a couple of years ago where you take a specimen and you rapidly freeze it in ice so that there are no crystals formed and you get what is called vitreous ice and so then you take an electron beam and you look at the structure and you look at density difference between water and your protein molecule and proteins usually have a density of 1.28 or whatever and and our name molecules of course are much denser they are about 1.6 and so they looked at the ribosome the mammalian ribosome and in the meantime this is an E. coli ribosome the bacterial ribosome and mammalian ribosome which is much larger the mammalian ribosome or this in this case it's from yeast that the eukaryotic ribosome is 80 s and the bacterial ribosome is 70 s and this is small seven in the large sub unit and the green that the green molecule there is a tRNA molecule that is that is in this case sitting between the two subunits and this is at a resolution of 11 angstrom and this is at a resolution of 15 angstrom so the surface features here are much pruder than they are here but what you can see is that what we have shown here is that the the eukaryotic large ribosome and subunit and small ribosome and subunit have these dark regions or these purple regions attached to them and what we found is that the eukaryotic large subunit and small subunit are exactly as the prokaryotic as the bacterial small subunit in its in their structure all what they have done is they have added additional RNA segments which are all found on their surface in evolution in these 3.5 billion years of evolution you have just made the ribosome more complicated and you added more RNA segments and more proteins and they're all found on the surface you see in purple here they're all found on the surface and in yellow here on the surface of the small ribosome and subunit and here's just tilted the whole thing by 90 degrees so that you see both surfaces and you see they are in fact in clusters on the surface here we have taken the small and the large subunit apart and you see the peptidility on a moiety and here this is a small subunit and it is exactly in the interface between the small and the large ribosome and subunit and here you can see again in purple is the eukaryotic ribosome with all its additional RNA segments and protein segments which are not present in the prokaryotic ribosome and here you may not be able to see it but here are these so-called expansion segments of the small ribosome and subunit indicated in red so the basic structure of the RNA of the ribosome and RNA is still the same what is different here is there are these expansion segments these loops and they're indicated in red and this is what you have then in the small ribosome and subunit we have then taken the RNA of the bacterial RNA of the small ribosome and sub and fitted it into because you can separate the densities of RNA from protein in these images because the RNA density is 1.6 and the protein density is 1.28 so you can separate the densities and you can fit in the bacterial RNA structure from the x-ray structure into these into these densities and you see that there are certain regions here and here that are that are not present in the bacterial ribosome and it is those regions which have the expansion segments so here for instance you have an expansion segment here you have another one this is unfortunately a mistake it should be yellow up till down here and there is another expansion segment here and it's just rotated so they're all on the surface and then we have been able to fit in the protein x-ray structures that are known into the protein densities and see we have been very successful of in the ribbon model if it's only 15 extra resolution to fit in into these densities the protein molecules and you will say well why do you do this exercise maybe in 20 years we have crystal structures of eukaryotic ribosomes and then this is all superfluous but it will probably take a long time to get crystal structures of eukaryotic ribosome but one never knows so we now have some structure that tells us a little bit before we have the x-ray structure and here's the same exercise with the large ribosome and subnet in red you see all these extra segments that you find in eukaryotic in the eukaryotic large ribosome and subnet and here we have fitted in the prokaryotic RNA and you can see the many empty spots here are the extension expansion segments that are filled in in color here by the expansion segments that are present in the eukaryotic RNA and then we have done the same thing with protein we have fitted in the protein models in this way now the next so this this is really a tremendous advance in in in the structure of this most complicated machine that nature has developed namely the the ribosome which translates the messenger RNA into proteins now the next question that we were interested in and this is also it will be in another paper published in cell next month is the channel we have isolated the channel from the membranes and we have bounded back to a ribosome which contains a nascent chain with a tRNA still in it and you can see the peptidyl tRNA here in green you cannot see the chain because it's the resolution is not high enough and then we have bound the protein conducting channel to it and you can see here's a small sum in the large sum and here's a protein conducting channel attached now we have rotated this a little bit so that you can see the channel from the bottom side in the large ribosome and subunit and here you see an inactive ribosome where there is no peptidyl tRNA and you can see an empty spot up here and the channel still binds but if you look at the conformational changes that the channel has undergone you can see an oblong gated channel binding to an empty ribosome and as the channel is penetrated by the nascent chain it rounds up and becomes thicker and so there are tremendous conformational changes that take place when the chain it goes through the protein conducting channel now here is a cut through these images and you can see the large subbing and the small subbing and the peptidyl tRNA of course you don't see the nascent chain it's just indicated here and here you see the channel and you see the various attachment sides of the channel to the large ribosome and subunit what is very interesting is that the channel is rather compact we don't see a big hole in the channel and this is the the miracle of this channel design because it is designed to to sort of cuddle the nascent chain as it goes across and and and not allow any other ions to go across or anything else across so it's very specifically designed to let the nascent chain in an unfolded configuration get across the membrane now what we also found is something interesting when the sex 61 the channel binds this particular segment of RNA it's a huge RNA helix swings over by 90 degree to get out of the channel of the channel exit side where the chain comes out this little dimple here is the side where the chain exits the large ribosome and subunit and this helix of RNA is protecting this exit side now when you bind the channel this helix moves by 90 degree parallel to the small subunit binding side and you can see this here and now freeze the freeze the this side in this case for binding of the channel so there are huge conformational changes that you couldn't see in a crystal structure unless you have a crystal structure of an empty ribosome and a crystal structure with the membrane protein sex 61 and this is not very likely to occur very soon and here is going into more details and analyzing what the connections are and I won't go and bother you with this but just you know the enormous amount of information yet you can get we have been exactly able to identify which of the large ribosomal proteins RPL 19 for instance and which residues for instance proline at 25 to 348 interacts with what helix of the RNA and so on so we have been able to do really some molecular dissection and so this this channel interacts both with RNA ribosomal RNA with ribosomal proteins you have also then fitted in the alpha helices in this channel and it turns out that three six 61 oligomers form this channel so it's very much like what we had drawn as a fantasy cartoon in 1975 20 years earlier that there may be three summaries of course it's entirely luck and anyway so now in the last five minutes or so I would like to talk yet about another of these public protein translocation systems the system that I just discussed with you that getting proteins across the ER is accessible to any protein that has a signal sequence you can make if you can take a a bacterial protein and and put a signal sequence on it and it goes across the endoplasmic reticulum of the of the eukaryotic membrane so it is it is that ticket that you need and the rest of the protein doesn't matter unless you have a sequence which opens the channel later really which is seen by the channel and then you become an integral membrane protein so this is one of the systems that I have discussed namely the endoplasmic reticulum but there are many other systems because there are many other compartments to which you have to send proteins you have to understand that proteins only live for a day or a couple of days and some of them live only hours and so they have to constantly be have to be resynthesized cells live much longer the proteins have to constantly be resynthesized and actually when they're slightly damaged they're not repaired like DNA when DNA is damaged there's a repair system to repair the DNA but with proteins there is no repair system there are little machines which take a damaged protein and and swallow it they look really like little dragons they swallow it and they spit out the amino acids and the amino acids are being used again so there's a constant turnover these proteins so these targeting processes have to go on all the time this is also true for membrane protein so you have to constantly make the membrane snow you have to make your mitochondria new everything has to be constantly renewed and so the other system that I want to briefly discuss is how things get in and out of the nucleus I briefly mention it to you and I will tell you why this is very important incidentally before I leave the endoplasmic reticulum I'll tell you what the people may say well what is what is important about that why why is this why did the Stockholm bother to you know to give a price for that I don't know why they bother but in the the it is it is what it has had great industrial use there are now proteins produced in the united states alone worth five billion dollars that erythropoietin is one of them which is a secretory protein which has signal sequences attached and use this mechanism to get out of bacteria and therefore you can purify them very easily then of course there are many diseases where this process of targeting doesn't work imagine the receptor that at fisher was discussing this morning if you have some problems with properly integrating the protein into the membrane that you will not have a functional receptor so there are many diseases and more and more are discovered where where individual targeting problems exist now here is proteins getting in and out of the nucleus and the reason why this is so interesting you have all heard about Dolly Dolly is taking an x-cell taking the nucleus out of the x-cell that taking all the genetic material out of the x-cell except for that that is in mitochondria and then fusing this enucleated x-cell with a highly differentiated cell of Dolly's mother in this case that then brings in the nucleus of Dolly's mother into this enucleated x-cell and what happens then there is a dialogue between the x-cell cytoplasm and the nucleus of Dolly's mother's cell that has entered this cytoplasm 1 million transport events per minute and after 12 hours of the Dolly mother nucleus in the cytoplasm you have gone back in the chromatin organization of the dna to that of a totipotent psychotic cell nucleus and so this is really an incredible thing it's an incredible finding we nobody ever suspected this would be possible have been done in frogs and it can be done in very primitive organisms but it wasn't it nobody thought it would ever be possible in mammals and Wilmut this demonstrated this in this experiment so what one can conclude is that the x-cytoplasm has all the goodies to reprogram a nucleus from a differentiated cell the working hypothesis if we talk about what can we do in the next 10 or 20 years is with all the ethical problems that we have with x-cells is that how about taking an in es-cell an embryonic stem cells which we now can grow in culture and hope that its cytoplasm when you take out its nucleus when fused with another nucleus from another differentiated cell of you or me would then become a totipotent es-cell of me or of you which would avoid all the ethic problems we cannot make an embryo out of that but we can potentially grow any tissue cartilage heart muscle anything that we want from these cells it will take a while until we understand how the es-cell differentiates I mean huge problems involved trying but not trying to to simplify things but we would essentially if this principle this concept works that the cytoplasm of these cells has really the information to reprogram the nucleus and the cytoplasm is a printed the components in the cytoplasm of the principle determines how many they are we don't know it could be five components excuse me five components it could be ten components it could be a hundred components but we have to find out what they are so we have to learn something about this traffic in and out of the nucleus and this is a problem that we have worked on and I wanted to briefly tell you where we are on that and here you have these maybe I go back you have these so-called nuclear pore complexes and they are huge structures the protein conducting channel is a miserly little thing of 220 000 daltons and molecular weight it's not very big this structure is bigger than the ribosome it's 25 times bigger than the ribosome it's one of the biggest structures in the cell consisting entirely of proteins so this is called a nuclear pore complex and all traffic in and out it goes to this nuclear pore complex and unlike in the protein conducting channel in the er where the chain has to be going across to a very narrow gauge and has not must not be unfolded the the the nuclear pore complex is more like a sewer pipe it lets a lot of things too but but it is very selective and how it is selective is really the interesting thing you have the same theme you have signal sequences you have signal recognition factors but you have a completely different transport principle and this is very much like sort of the Bach Goldberg variation it's a simple theme signal sequences recognition targeting translocation across a poor channel whatever but the variations of the scheme of the theme are really so exciting I mean I wish I would have time to talk about mitochondria and chloroplasts and peroxisomes they are in each cases the theme has been varied in the most fascinating ways not to talk about bacteria we heard from from Edmond Fischer today that bacteria have the Yersinia the pest causing bacteria has it on its plasmid coding for a phosphatase which is a really a eukaryotic protein which the bacteria then secretes through a needle which it polymerizes from one single protein one single protein of the bacteria polymerizes a long needle with a narrow gauge and this needle while polymerized punctures the eukaryotic cell and puts this protein that Edmond mentioned from the bacterial cytoplasm across three membranes two bacterial membranes and the eukaryotic membranes and injects it by this molecular syringe a real nano device if you like nano technology injects it into the eukaryotic cell this is just bacteria of the absolute masters in this and we have very far from understanding things but let's go back to the nuclear pore complex so if you look at the nuclei by free structuring you see on the surface of the nuclear membrane you see these pore complexes they're actually very beautiful structures you can see an eight-fold symmetry they look like flower structures and here is an image that we have done that shows you the pore complex there is in the center in pink here this huge open pore it's 25 nanometers when it is open for those of you who know the nanometer world it's a very very big opening and then there are all of these fibers and spokes and wings which connect this central tube to the membrane it's a double membrane there's a hole in the double membrane and this whole structure of the nuclear pore complex is anchored in the structure now so i'm going to rotate it for you a little bit so that you can see what it looks like see there are fibers here and there's the center structure here the two membranes and if you cut some of the membranes away you can see that there are there's a nuclear basket and then there's fibers which point to the cytoplasm and so you can see this we have cut it away again let me stop it here so you can see the membranes have been cut away right and there is a circular opening in this double envelope membrane in the center is this tube through which all the traffic goes and here are these cytoplasmic fibers which go into the cytoplasm and they are like the like the tentacles of a jellyfish they are roaming around and if you have a signal sequence to go into the nucleus and the appropriate signal recognition factor you can dock on these fibers and so what happens is that substrates which have access to the nucleus are first concentrated there and then by a process of probably diffusion they go across this at the channel and there are these two structures which i come back in blue is the is in so-called nuclear basket and here are these fibers which are you find only on the cytoplasmic side in blue you find fibers baskets only on the nuclear plastic side and this is my grout slab has just been able to identify all 30 proteins by a proteomics project and localize them to the nuclear pore complex but i won't go into this now and i will be very short too so a substrate which is indicated here in pink will be recognized its signal sequence will be recognized by a signal recognition factor it will then dock here on one of these fibers and then it will it can go across it can go in either direction force it or back and then it binds this high affinity to one of these blue fibers there and then there is a small gtpa which dissociates and this makes it possibly irreversible and when you export something it's just the opposite you assemble something which contains an export signal with a transport factor and with the small gtpa is you dock and then you diffuse across to the other side and then gtp hydrolysis gets you into the cytoplasm I mean it's a very quickly summarized what has been worked out in the last five years or so in in one minute but it doesn't really matter whether you understand these principles in great detail just let me show you another little cartoon which made a sort of the take-home message what this would look like trying to get the little hand here into the picture to wait until it comes or here it comes so now you have the membrane the outer membrane the inner membrane of the nucleus here are these the central pore is not shown here here just the fibers on the cytoplasmic side and on the nucleoplasmic side and what we imagine is that these fibers move right and so what you now see is is the movement of these fibers and now comes a hapless protein along which doesn't have a nuclear localization signal and it just doesn't get there because the fibers entropically excluded now comes a substrate which has a which has a transport factor it can bind to the fibers it goes across docks here and then comes a small gtpas which dissociates and it can go in i mean so this is a very different mechanism then you have seen for the translocation of it unfold the chain across the endoplasmic reticulum now and i have just one more two more slides and then we are free now here is a picture that has been done some time ago by Ron Lasky's lab in england in cambridge and what he has done is he has taken gold particles these these round particles or gold particles and he has coupled them to a nuclear protein that has a nuclear localization signal and then he injected this into the ooplasm of an oocyte and this is an electron micrograph and the preservation of structure is not very good but you can see the nuclear envelope here and here's a poor complex here's a poor complex here's one and here's one and you can see then how these gold particles dock on these fibers in the cytoplasm you can see how many there are we have calculated there are sites for more than 500 docking sites on these fibers so that you have really a zone that concentrates your substrates that wants to go into the nucleus first before you actually go into the nucleus and then you go you can see that they go single file this is now the other side they go single file across the nuclear poor complex into the interior of the nucleus and what has been found is that there are actually filaments which extend from these nuclear baskets of the nuclear poor complex into the interior and they branch and they form some sort of intra nuclear subway network because the nucleus is very crowded i already told you two meters of dna in 23 and 46 pieces in our case and and so it's very crowded and so you you have to create a subway system that you can go very very quickly by diffusion and you're not hinted and these fibers create a chromatin free space in which this very rapid transport can occur to the center of the nucleus and recently we have been able to isolate this here i just wanted to show you you look by an electron micrograph from hun threes you look at the interior of a nuclear envelope and you see one poor complex here another one here and you see the nuclear basket these nuclear basket fibers and here these filaments these branching filaments have been ruptured of you see just a little blob here you see a little blob here but here you can see they have been they have not been ruptured of and you can see how they how they are most and how they branch and they go all the way to the interior of the nucleus and they create this chromatin free zone for this intra nuclear subway and and this of course is there must be entries and exits and so this is a new research area very fascinating and here we have isolated these fibers you can see they of course they have aggregated on the grid and so they are we are just in the beginning to analyze them but what is already stunning that these fibers are made from a large number of alternatively spliced forms messenger honest of the same gene and what we expect is as you expand the chromatin you have like in a lego set you have proteins which have shorter or longer fibers portions and they can then interact with the chromatin surface of the expanded chromatin and then can create this chromatin free space on which you can then build other molecules of course there are many other molecules which interact to allow facilitated diffusion so i have given you in this in this too long lecture a short overview of how exciting the field of protein traffic war and how is and it still is we are very far away from understanding it and what the practical implications are now remember if we would understand what goes in and out of the nucleus in the case of dolly if he would be able to isolate the molecules we could essentially make the essence from any of you i mean there's few other things you would have to engineer and we could essentially grow any tissue that we need to replace in you we couldn't make an embryo so we but we would avoid all the ethical questions which which have plagued us over a very long time and we would avoid we would sidestep them and and this would give us of course a tremendous potential to repair many many diseases by cell therapy we wouldn't have immune rejection it would it would really be a wonderful thing and and it will take a long time until we find out but it is one of the applications of studying communication between the nucleus and the cytoplasm thank you very much we'll uh gather our panel for one last time for a few questions for dr. global i have a little bit of water on my hand thank you thank you thank you thank you it was a little bit superficial perhaps but you know for the audience i had a few things that were published and again it's just a ribosome story you can go and talk for an hour the guy said he heard the music already he still said well i mean the really key advance was the crystallization of the color ribosome yeah archaic ribosome but uh dr norby will be joining us soon yes the marginal man i have a question for dr. global to begin with here if the data that you're showing us is unpublished does that mean that it won't be on the test no it will be published in one month in two papers in cell but one thing that i wanted to add particularly for the young people i wanted to give you the feeling how much there is to be done just mention think of the example of dolly uh we have to understand what are the factors are five or ten or fifteen how far we have to go and i hope that many of you will join us in this fun it's a tremendous amount of fun you can see i'm still excited about these things even so they happened 25 years ago because they are beautiful constructs and uh so you will be compensated by low pay but by a high degree of high of excitement let me begin with uh asking for comments from the panel anyone dramatics in another context einstein said god does not play dies um in this case it seems that you've been saying nature leaves nothing to chance inside itself what the impression you gave me everything that produced is guided to the place where it's needed by some inbuilt mechanism is any room therefore for talking about diffusion should we say in a cell as the product of mass action and uh exaggerate in what i said at the beginning but really what is the balance between guided placement proteins and just sheer diffusion ultimately in the case of nuclear transport it will be mass action but it the diffusion will be facilitated because on these filaments sit a number of uh nuclear porans um that are not only in the nuclear pro complex it serve as stocking sites and uh these nuclear ponds can interact with transport factors you have bigger leaps than you have getting through the dada nails of uh or this straights of sizzling that that is really what the nuclear pro complex is but then you have um facilitated the diffusion also within this chromatin free zone that is delineated by these fibers uh we think when we don't have any proof yet there could be other fibers that involve that are involved but um we think it's only those fibers okay dr crodo what's the structure of this syringe that you described as a nano thing oh that what what's the what's the little syringe that yasinia has made has probably the most has been the deadliest weapon ever assembled in in in history because it has killed more far more people than uh than the AIDS virus and this little syringe is a pro is made of a protein that is 6000 in molecular weight and it can it is polymerized in the inner membrane of the yasinia bacteria and then goes through a channel in the outer membrane and is polymerized we don't know how into a spiral kind of of of syringe with a very narrow gauge i don't know what it is uh pen angstrom not more than that and and so the protein that gays goes across must be unfolded how the how this uh phosphatase that you mentioned is then unfolded and it's selected to go across we don't understand but it is we have published that you can see it in in pna as that the syringe is absolutely magnificent because you can see it in negative staining you can see these beautiful needles and you can see the center of the needle of course empty because that's where the protein would go across so it's a nano it's a nano syringe if you wish Dr. Fisher any idea how uh axons go to their target in the brain do they follow actin corridors uh type of subways like you you mentioned in the nucleus any idea about that because they go you know centimeters yeah well but that will be probably the question was how how do axon and dendrites connect with each other i talked of about 10 000 connections of each neuron in the brain some of them have more some of them have less and how do they find each other the numbers are staggering i think it's 300 000 synapses per second that are formed so if you have a child 300 000 synapses per second when it sees something it has to integrate all of this information by connecting the prop the nerve cells with each other and so what happens exactly you know they do very simple models one can propose that these are integral membrane proteins that are highly localized in a in the dendritic portion of the plasma membrane and then via this concentration there uh and again mass some sort of mass action and then of course there are factors that have been isolated and so on and so on and i'm really not working in this field and i don't want to trivialize it i mean it's it's there's a tremendous amount of work going on where there are gradients of of of soluble proteins which help then in in attracting uh a deformation of neurons any other questions from the panel here have a question here if the body rapidly catabolizes proteins what is the functional lifetime of therapeutic proteins well um what i was talking about are proteins in the cell and in this cell there is a magnificent looking machine which really looks like a dragon called the proteasome in this proteasome eats damaged proteins and the proteins are first marked for destruction by putting a little ubiquitin molecule which is another short protein to it and this is a very important discovery because protein degradation plays such an important role in in cell control i i i don't want to go into that but so these are machines which have been developed for intracellular degradation now proteins which you get give from the outside they can be endocytosed and then they can be degraded in the lysosome for instance we heard from prusina today that if you stimulate perhaps endocytosis that these proteins the prion proteins may have a better chance of being degraded in the lysosome perhaps i mean maybe that's one of the um consequences of this but um it it depends um um how not how the intracellular machinery can degrade it but this machinery that is there in the lysosome in the endosomes that degrade or that are on the surface of the of the cells membrane proteases on the surface of the cell or that are secreted how resistant are these proteins and erythropoietin for instance is not is not uh lifetime i don't know it probably a day or two something like this half life half life so you have to inject these proteins over and over again so another one from the audience here is there a way to attach a signal tail to a protein without one so that it can enter the nucleus yes you can genetically engineer any protein that you want and you can put the right signal on it and if you do that the protein will will go where the signal leads it to so people have taken proteins and got them into mitochondria or into chloroplasts or into the nucleus or make make a cytosolic protein into a secretory proteins so people have done this so you can you can essentially do cell engineering if you if you want to do that okay here's another technical one for you with reference to the dual membrane in the mitochondria how can a single stop transfer signal be read and integrated into the intermembrane after that same signal was ignored by the previous translocon of the outer membrane now that's a very interesting question which has not been solved yet I would imagine that the channel in the outer membrane reads a little different than the channel in the inner membrane but there is not that much information yet we have to be patient here's one little less technical how did you develop so much enthusiasm for your subject I've always been very curious and I've always liked to to to make working hypothesis and I've done quite a few of them which were killed by ugly facts and that that has never discouraged me and it's it's it's nice to have a working hypothesis and then if it fails while you say well it's just another hypothesis killed by ugly facts but if it if you succeed it gives you a very wonderful satisfaction that cannot be surpassed by anything else at least in my case one more technical question here says inhaled proteins are readily absorbed through the lung does the lung have a protein conducting channel which is analogous to the ribosomal system this is well the lung epithelium may not be may not be totally closed all the time and this is why when you the connections may may be leaky and this this is the way it would get across I mean this would be my explanation but there are more people probably in the audience who can answer this question better I'm not a lung epithelium epithelium specialist okay another question here what aspects of proteomics do you see that might be most useful for practicing physicians in the coming years well I mean there is a very interesting thing people are looking at proteins in the urine and they are very sensitive machines now the new spectrophotometers can detect auto modes which is almost we're not yet at avogados number of single molecules but they were coming very close and so it's it's worse to look at urine again and urine is of course the kidney is a filter in many proteins almost all proteins get across and then they are reabsorbed and so the urine is a wonderful diagnostic tool and going from the early physicians who dip their finger into the urine and put it on their tongue to see whether there was diabetes whether there was sugar in the urine we have gone a long way and it is worthwhile to look at the urine I was just at a biotech company meeting where people are actually looking at the urine and it's amazing the number of proteins that you can see and that you can actually use it for diagnostic purposes and that will be a huge new frontier where you can just take a drop of urine and you can you can tell what because each disease has its own footprint leaves its own footprint in the blood and leaves its own footprint in the urine eventually and if you have highly sensitive matters you know it's just a drop of urine you can tell that's what you have okay do we have any other questions from the panel one closing question here from somebody who remembered Dr. Croto's talk from yesterday uh did you have a McConnell set when you were a kid no I was I was not I was not one bit interested I mean I would totally contradict Harry what you said I have was not one bit I was only interested in architecture maybe I mean I I wandered through this beautiful old medieval city that I was privileged to grow up Freiburg near Dresden which was a complete intact medieval city with with city walls and magnificent gothic and renaissance building and I choose my my my ways with the city each time to see another building another street and I would go in these buildings and look and I was very romantic and I was very impractical I couldn't my father said you were so impractical you only can be a lawyer you cannot put you cannot put a nail in the wall but you can argue for hours so there wasn't I haven't really played with machines but on the other hand didn't become a physicist that didn't become a chemist and I think there it's probably much more required right it's just this guy to fix your car speaking of that who was the architect was it Palladio because it has the solidity and the Greek features of a Palladio your your Dresden church no the Dresden church was built by by George Georgi Bair who and it was built between 1723 and 1742 and it was inspired by the Italian by Santa Maria della Salute in Venice even so Bair had never been there when August the strong of Saxony traveled to Italy he he said you know I must have in Dresden a cupola church and of course you know once he demanded that you know the architects immediately went to work and built this and Georg Bair built this cupola church but he didn't didn't want to build a cupola church just like any other church so he built a cupola hundred meters high you have to imagine in form of a bell so it is called the stone bell or the Steinerne Glocke and this is what the absolute unique thing of the Fraunkirche is it's the only church which has a stone bell the Steinerne Glocke and when it is finished it will be one of the ten world oneness again and this is why we all very enthusiastic the interesting amongst our panelists here are our Nobel Prize winners we have a musician a graphic artist an architect and a poet down in the end let's thank our panel for an absolutely wonderful discussion so we've had we'll reconvene at 6 30 this evening