 Now, I think I'm going to say that there is some kind of a lecture here. There has been a lot of service in the department for over 50 years. They have put a good impact around here. You have this little brochure that tells you something about there. It has a very nice picture. It actually looks like a picture there. Where he came here as a student and stayed on the staff. And probably the greatest part of the mission was the demonstration of the radiation with active vitamin B. And this observation mainly virtually wiped out where it was in the world. Those are tremendous advancements. Also, the commercial application of vitamin B and radiation was obvious. And so, the system was patented. And here we wanted to give this so that the medicine front of the department could utilize the foster research around the university. He stricted, he was a grad school, and some other businessmen suggested that they start a foundation. And so, this was the origin of the commercial and the research foundation. Everybody wanted to give all of the revenue from the patent to the war foundation. But they suggested that in the future there won't be some poor, destitute, vile kingpaw. Who would want to have some of the funds from the patent in one day, way of years. So they insisted that they would take 15% of the patent money. And the rest of the way, it was a commercial and research foundation. They would take that 15% and make a grant to the grad school research committee, which would then distribute the funds around the campus. A few years ago, 94% of the funds coming to war came from patents that originated in the College of Department. But this doesn't mean 94% of the funds coming back to the biochemistry. Traditionally, 12% of all 5% of these funds came from the graduate school committee went back to the biochemistry. The others were the students of war. In this day and age, you can imagine how much impact those war funds had here in our early research at the University of Wisconsin. This was before funds were available from the NIH, NSF, and so forth. National Science Foundation was starting in 1950. At that time, they had $3.5 million for all types of research nationwide. That now has gone up not 1,000%, but 1,000 full since those days. Now, back before the NIH and NSF funds were available, the funds from the Wisconsin University Foundation sponsored the funding for graduate students and such, and really had a tremendous impact to give the University of Wisconsin a real advantage in the recruitment of those days. Her contribution did not end here. Ian Elvin Steenbach gave funds for many types of arts and humanities around the Madison area. You'll find a dominance of the Madison Symphony Orchestra and many other things. And some of the roles he did from his bill went to the Wisconsin Academy of Arts and Sciences and made a tremendous difference to the program over there. So, Herri and Elvin Steenbach had a tremendous impact here on the local community and more specifically on the University of Wisconsin campus and the support of research. So, we recognize Herri today through this Steenbach fellowship. Yes, they're global as they help to me as usual. Bob, I want to thank you and the department for allowing me the privilege and the honor of introducing Dr. Gunford Lowell as the Herri Steenbach two-day lecturer. I knew Herri Steenbach and he was on my thesis examining committee just 59 years ago. It's nice to have former graduate students and post-doctoral fellows come back to Madison for a lecture. But it's especially gratifying when one comes at the invitation of one of the MacArthur Labs sister departments and when the reason, the occasion is for the most distinguished lectureship in the natural sciences, the Herri Steenbach Lectureship. In terms of recognition for an outstanding career and research, Yes, they're global is undoubtedly the most outstanding person coming out of the MacArthur Laboratory in terms of the recognition that he has received here in the United States. His record is in the leaflet that's already in your hands. Then I shall not list all of the honors that he has received there in print. He came to me with an MD in 1962 and worked toward a PhD the hard way with much required coursework. What the record doesn't show is the story of Günther's flight from the eastern part of Germany as the Russian army approached in 1945. They left their home as mother and her four other children and Günther left their home and found shelter with relatives somewhat west of Gresden. They witnessed the fire and smoke as that city was totally destroyed by a live aerial from Martin. Günther had been married for 20 years to the same lovely lady named Laura and he has used some of his prize money to initiate the landscaping at the entrance to the MacArthur building and will help with the restoration of Gresden's famous church. It's my great pleasure and honor to introduce Dr. Günther Wolff. Well, thank you very much and it was a lovely introduction. It was a very great pleasure being back in Madison to see so many more friends. It brings a lot of many memories, many sweats. The cream in his hand, the chocolate was fading. That saved me from fading in the last minute. And other very pleasant memories swimming in the lake at this point when I left in 1962 you could still see the bottom of the lake. A little bit later all of these weeds started growing and the weed harvest has been long. So I thought I would, what happened to my screen suddenly off? I would like to show you a arm just touching it. I would just like to show you a picture of Van how he looked in those states. I wrote the song. So now I go down and click on it. So this is Van sitting at his desk. At this moment in his life he was very interested in Frank Lloyd Wright in the Monona terrace. And as I hear the Monona terrace finally is being built. The van in those days in the early 60s was very much interested in having the Monona terrace constructed. And his efforts have now come to fruition. And it's very, very nice and lovely to see. He also then became interested in diurnal variations. And you see him here involved with all of his papers. And he used to go into his office to announce our new findings and we would have discussions with us on this. Now Van mentioned Dresden and I just thought I would show you a slide of that. Let me see whether I can see it. This is the skyline of the city which was considered one of the most beautiful skylines in Europe. And you can see all of these rock or core and the rock towers. And the most famous of these is, let me see this. Is the Frouenfeldt, this one. And all of this has been reconstructed except for this one. And I have founded a foundation called Friends of Dresden to collect 10 million dollars. And we have so far one million which is not bad to help in the reconstruction of this church. I also show you the church in its full glory here. The particular nice aspect is its cupola which is not just a semi-sphere but is in form of a bell. This is over 100 meters high. So it's a very large building. And the cupola is in form of a bell. You can see here it's a bell shaped thing. And this was very unique. It is really the only bell-like cupola and it's also called the famous stone bell. And it is going to be reconstructed exactly as it was. So if anybody of you is interested in helping out, I'm available for contact. So now I don't want to talk only about my biggest life in what I do besides science. Incidentally, this reminds me a little bit on the nucleus. Tomorrow you will see nuclear pores in these little windows. Anyway, so what I would like to talk about in these two lectures today and tomorrow is showing this high-resolution microbar itself. What you see here are some of the principle organisms. You see here the nuclear envelope, double membrane, and you have these 100 nanometer pores which are filled with a pore complex. And we know that traffic in and out of the nucleus is bidirectional. Proteins go in but proteins come also out. P's go in and P's come out. DNPs like viruses, adenovirus, for instance, also go in to the nucleus pore complex. So this is a bidirectional, very large transport organism. And I will talk about this tomorrow. The other major transport system which I started out to work with is the endoplasmic reticulum. Incidentally, I was just reflecting on the last 30 years of my career. Actually, everything I've done is I've started here in Van Pottislaup. We started to work on the nucleus and we developed a procedure which isolated the nucleus. Van, you may remember, you wrote the paper actually. And it was a very nice procedure to isolate the reticulum in high yield. It was published in Science and I continued to work on the nucleus. And I also worked in membrane-bound ribosomes in the endoplasmic reticulum. And from the membrane-bound ribosomes, we then went through the signal health processes and through all of these other things which I will tell you about in the lecture today. So basically, I have continued the work that I have started in Van Pottislaup over the last 30 years. And as you can see, I'm far, far from finished with this work. And it's still an unfinished symphony to a very long extent. I shouldn't even say symphonies. But in any case, so the other major systems are, of course, I don't want to go into detail on mitochondria where you have translocation across outer membrane and across postmembranes. And you also have proteins translocated from the matrix into the internal membrane space or integrated into the membrane. So there are three distinct translocation systems in mitochondria. Three distinct ones in chloroplasts. I will briefly talk about one of them at the end simply because it is so different from the one in the endoplasmic reticulum. Now, what all of these translocation systems have in common, those which are unidirectional and those which are bidirectional, is that they all have the information for this process is encoded in the protein itself in what we have called a signal sequence. And that signal sequence is then recognized by a couple of soluble factors which targets the protein to these membranes. And then you open up a protein conducting channel which allows passage of the protein across the membrane or in the case of membrane proteins which also have a signal sequence and also need to be targeted. You have an additional what we call topogenic sequence which helps in integrating the protein into the membrane which we have called a stop transfer signal sequence. So what I will talk about today is primarily how proteins get across the endoplasmic reticulum and also a little bit about this system because it's interesting. Now, again, the story of this goes back a long time and then it started in 1975 and we were able to set up a cell-free translation system. And that cell-free system really, I don't see that I can try it with a light arm because it may be not a protein sequence. So I have to go back to this and keep you in the dark. So in this cell-free system, face-fully we produce a translocation of secretory proteins into the endoplasmic reticulum. That is we were able to take a secretory protein, translate it into a cell-free protein synthesis system, add micro-sonal membranes and we got the chains translocated across the membrane. And that was really a key to set up this cell-free system because then it allowed to look at protein translocation from the biochemical point. Once you had it in the in vitro system, you could take a party in vitro system and you could study the various aspects of it. And so I'm not going to go and summarize and tell you the entire history of this but the first thing which we isolated was this so-called signal recognition particle which is indicated here. And what the signal recognition particle does surprisingly is an RNA-containing particle which contains 7 sRNA and 6-polypaptide chains. This particle interacts with the ribosome and recognizes signal sequences which emerge from some sort of space in the ribosome or subunit and then this is the process of recognition by the cytoplasmic signal recognition factor. And then what happens next is that the signal recognition particle, the SRP, then is recognized by a signal recognition particle receptor, SRP receptor in the membrane. Maybe I can focus a little bit and see what happens. Do this because it seems to be all out of focus. I cannot do it. So you see this is focused. This is a little bit better. Okay, so this SRP then binds to an SRP receptor and there is a number of reactions which have been largely figured out in Peter Walter's lab in San Francisco, in Gilmore's lab also and worked in our laboratory previously. And what they have shown is that the SRP has one of the six proteins of the SRP of 54 kilodollars is a GTPase and also binds to the signal sequence. And the other and the better subunits are also GTPases. So there are three GTPases involved in this targeting process. And I won't go and explain you precisely what happens here because many of the details are non yet. I just wanted to keep in mind that there are three GTPases involved in that. And somehow then the signal sequence is released on SRP and then if it interacts with what we had posted to be a protein conducting channel binds to the protein conducting channel and together with the binding of the ribosome and the signal sequence opens this channel and chains then translocated across the membrane. Now the protein conducting channel has been one of the most controversial aspects of what we had called the signal hypothesis. And it received bigger disopposition because it was thought that the hydrophobic signal peptide would just partition into the bilayer and then from the free energy which you get from this partitioning it would be enough to get the chain across the membrane. This was proposed by Steitz and Engelmann in 1980. And of course we had proposed this channel in 1975 and we were never able to get any evidence and as time went on I was going around and talking about channels and hand waving finally an electrophysiologist came to me and said why don't you send me some membranes and we will do some patch clamping and then we should see whether there is a channel. And so a couple of months later hesitantly I called up because I hadn't heard from this person and have you found this channel? No, I haven't found this channel. So it was very depressing for me that I sort of had almost given up on the idea that there must be a channel but in two years it sounded perfectly fine because you have channels for ions. And the polypeptide chain after all is an ion, a polyion. So it's unlikely that the polypeptide chain was charged residues with just reverse in the bilayer. But of course there are models which have been suggested like this one by Schatz for instance that a chain here indicated this positively charged residues in the case of mitochondria could just interact with a rearrange lipid bilayer like this where you have sort of a hexagonal phase transition and that the chain then could go across the membrane. So this was proposed in 1986 by Schatz. So the idea of a protein conductor channel was in fact not acceptable. And it was a very controversial idea. In fact I had one of my blanks almost turned out for dogmatic adherence to our loaded ideas. And so it was of course very difficult to persuade Sandy Simon who was an electrophysiologist who joined my lab specifically not to work in electrophysiology but to do cell biology to persuade him to look for this channel. And so what we did essentially we went back to a very old method. We just let me show you here the endoplasmic reticulum which you see the membrane started with ribosomes and when you homogenize the cell and the endoplasmic reticulum that breaks up into little so-called microsomal vesicles. The vesicles are sealing spontaneously and you get these microsomal vesicles with the ribosomes on the outside and each of these vesicles contains about 100 or so ribosomes, right? So we went to the very old technique of Müller and Rudin which had been published some time ago using a planar bilayer system. And the setup is as follows you have two chambers, sorry I don't want to frighten people off I'm not an electrophysiologist because I didn't know anything about electrophysiology until I met Sandy Simon and he taught me a bit about it and I still don't know much about it. In any case, what this system uses is a chamber about 3 ml of liquids on both sides and this is a plastic division between these two chambers and then you have a little hole in this plastic division and you can paint the planar bilayer on this hole. So this is a planar bilayer in this little hole here and if you have a voltage clamp and you measure current you don't measure any current because a little bilayer cannot conduct any ions but the minute you put a channel in this bilayer then of course you get ion conductance which you can measure by current flow. And so let me see whether I can focus this a little bit they seem to be all out of focus here. Now, so when you add rough microsomes with the ribosomes here in red to this chamber let's call it the cis chamber and you do it with a smart gradient you occasionally, really but occasionally do get fusion of a vesicle with this planar bilayer. And so when this happens you see occasionally a few channels of 100 picosiemens channels which are usually larger than ion channels but that's all we saw and that was very disappointing because we expected that we would see maybe a 100 channel each ribosome sitting on a channel and we thought we would see maybe 100 channels and we would see big conductances but in fact we saw only one channel or two and of course we wouldn't know what channels are they are they protein conducting channels are they channels to get ATP across sugar nucleotides which have to be transported are they channels for those processes or what are they? So then, of course the good thing was that we saw channels so we knew that we had some fusion but the bad thing is that we didn't see what we wanted to see, maybe 100 or so channels and so let me decide why is it that we don't see the channel could it be that the polypeptide chain which sits in the channel will not allow conductance of ions so why these ribosomes conduct a chain across the membrane here it cannot conduct simultaneously ions at least not large quantities that we can detect and that's what you actually would suspect because you know that the lumen of the yarn for instance has millimolar concentrations of calcium and the cyroclastic has only sub-numeral concentrations so the lumen of the yarn has segregated within different ions and if you would have a leak particularly a large protein conducting channel you would eliminate this gradient of calcium for instance so one would imagine that while the chain is going across the channel there is not at the same time co-conductance of a large number of ions so when the chain sits in the channel we will not see any conductance of ions in other words this channel is electrophysiologically silent cannot be detected and of course if the ribosome comes up and the chain has been trans- okay what could imagine that the channel closes immediately again for the physiological reason that you do not want to have act of liberation of calcium across this membrane so this is we figured it may have been the reason why this electrophysiologist would try a patch clamping they didn't see anything because the channel is either closed or when the chain sits in the channel it doesn't conduct ions so then we thought how could we trick the system that it would conduct ions and so one possibility is to kick out the nascent channel and keep the ribosome still attached to the membrane and a few years earlier with David Sabatini we had worked out a procedure where you can precisely do that at low salt 50 millimolar salt for instance you can add pure myosin and the pure myosin will then release the chain from the ribosome and will get it across the membrane for instance if you give pure myosin to tissue cartridges out these peptidyl pure myosins actually are secretive now many of the new graduate students here probably don't know what pure myosin is anymore than I do at work we also do in pure myosin and actinomycin in our experiments nowadays it's Gretelin and something else but let me quickly show you what the structure of pure myosin looks like it's really an analog of an amino acid tRNA with a nucleoside moiety here and an amino acid here and so if the tRNA of course you have a large number of nucleotides which hold onto the ribosome the pure myosin you do not have that so this reacts with the carboxa terminal of the nasium chain and the peptidyl transferase of the ribosome couples this amino terminal of this of this amino acid moiety here to the carboxa terminal of the nasium chain and since we have nothing to hold on the peptidyl pure myosin just slips out of the ribosome slips across the membrane and is actually secretive so when we gave the peptidyl pure myosin to the cis side of the membrane so we gave the pure myosin to this side of the chamber the pure myosin needs the ribosome to couple it to the nasium chain if you give the pure myosin to this side nothing will happen the pure myosin cannot cross the membrane and therefore it will have no accessibility to the ribosome and therefore nothing should happen so we added the pure myosin to this side and lower and behold what we sorry I'm going backward here lower and behold we saw a huge increase in conductors we see the pure myosin is added this is now in the minute scale and we see a huge increase in conductors this is now a sequence and so since this was all done in low salt these channels remain open and the ribosome is still attached to the membrane and this is of course an unphysiological situation normally when the nasium chain is released from the ribosome the ribosome problem comes up in the membrane and the channel is closed but in this way we have tricked the whole system and the channel remains open and we get this huge increase in conductors now the next experiment of course when this somebody started in the lab needing some of his competitors he immediately said well this is an algorithm because you know it's clear the pure myosin is only 95% pure and there are all sorts of other ionic force in there and all what you have done you have added a bunch of ionic force and now you see a huge increase in conductors but remember that we have this very nice control that we can add the pure myosin to the trans and then it shouldn't do anything and in fact this is what Sandy did when he added it to the trans channel where the ribosomes are not exposed you see you see nothing and then you add the pure myosin to the cis channel at this point you see again this huge increase in conductors in this case we got up to 16 amosemes so this was at least decided this of the membrane quickly used as a very powerful argument that what we are observing is the true effect the pure myosin really cleared out the channels and therefore these channels which normally are not designed to conduct ions but they are designed to conduct the polypeptide chains now are able to conduct ions under voltage current conditions and actually the conductors for those of you who have prism in electrophysiology 62 million ions per second ion channels usually have a couple million ions per second so it's a huge challenge of course we cannot estimate from this sort of rather than diameter of the channel but if you assume that a chain is going to the channel in a loop configuration it must accommodate two parallel, anti-parallel loops it could be as large as 20 angstroms in diameter ok so the next thing which Sandy did is to add pure myosin at very low concentrations so that perhaps you would clear one channel after the other and we could see individual channels and so that was really very nice you can see the added pure myosin in sub-micromolar concentrations and you can see now that nothing happens for a while and then one channel is cleared and we see a drop of 220 picosiemens at this point so one channel has been cleared this channel stays open and then nothing happens for a while and then two channels are cleared actually if you do this in seconds you can see a nice discrete is a distinct step in between and then a fourth channel was opened and so on and we have now recordings where we can go on for a very long time the limitation in these experiments is that very often your membranes break and then your experiment comes to an end it's a technically very demanding procedure so it is a procedure which takes a tremendous amount of patience but anyway so these distinct steps then indicated to us that in fact each of these protein-conducting channels has distinct conductive properties in that it can conduct up to 220 picosiemens worth of ions okay so then this is all I'm going to tell you about this channel and there is another question which I really want to discuss with you and I will come back to the channel a little later on because it has been isolated primarily by genetic approaches primarily in Randy Schekman's lab but I would before I go into what the channel looks like raise another problem with you and that is how do membrane proteins get in and what we postulated some time ago that membrane proteins of course do have signal sequences as well and we showed this in the case of a protein of a virus the syculestomatitis virus like a protein which is a transmembrine protein with an amino terminal on the trans side and a carboxy terminal on the cis side and we showed that in fact it does have a signal sequence and we showed that this signal sequence competes with secretory proteins for transportation across the membrane so the sequence of the membrane protein of the secretory protein are addressed to the same transportation system in the ER so what happens then we postulate also we don't have any evidence at this moment is that another sequence which is hydrophobic in nature is able to open the channel laterally to the lipid bilayer so that this so called stop transfer sequence can be displaced from the channel and chain translocation subsides and the carboxy terminal of the chain remains in the cytoplasm and of course the channel closes and what we also postulated is for membrane proteins which span the membranes many times that you can repeat this maneuver you can open the channel again and you can integrate the next segment of the channel so there is other models of how you can imagine this that maybe that you can actually integrate more than one loop into the channel by recruiting more members of the channel proteins or by perhaps recruiting heat shock proteins because the membrane is integral membrane proteins I don't want to go into this because it's all speculation and there is very little data on this but I just want to keep you aware that membrane proteins use the same mechanism of a signal sequence but then somehow they escape a segment which passes through the channel and escapes from the channel so this protein conducting channel is more than just a passive conduit for polypeptile chains it is really a proof reading it's something which reproduces features of the LAC chain and can therefore do the opening and closing in two dimensions ion channels don't do that they open and close in one dimension so the protein conducting channel is different from ion channels in a way in that it can open and close in both dimensions so now I want to talk a little bit about unfortunately you won't see anything here this is briefly summarizing what this channel looks like there's much prettier pictures which have recently been published by Chris Ake and Chris Miller and Tom Rappaport who have isolated the channel complex now just very briefly the history the proteins of the channel complex was first described in Randy Shackman's lab by genetic means and then Peter Walter added the components to the system namely sex 71 and sex 72 so the components of the channel is sex 61 alpha beta gamma 62 and 63 we are identified in Randy Shackman's lab 71 and 72 in Peter Walter's lab in yeast and Tom Rappaport has identified the components in the mammalian system and these proteins in this case are isolated in our lab here by Ronan Beckman using a tag on sex 62 which can be cleaved off with a factor 10 site and you can in one step purify in commasistane quality this channel and you can put it on a plate and you very see these donut-like structures which are not as well visited here as the one which you have recently seen in a paper by Chris Ake in a seminar in Tom Rappaport itself of course if you look at negative staining you can see very often stain depositing in these central cavities or whatever large proteins may have a dilute where negative stain deposits that doesn't mean necessarily that this is a channel so what we have to do is we really have to have three dimension reconstruction on a channel and what Ronan Beckman is now doing is binding this to the ribosome and then it's doing vitreous eye selector microscopy three dimensional reconstruction and that should really give us an idea whether we are really looking here at a channel but anyway and then of course very much like in the island channel where we have to figure out what is the signal sequence binding site, what are the ribosome binding sites, how is the channel created open across the bilayer channel open to the bilayer and so on so there is a huge amount of structural work and biophysical work which has to be done in order to understand how this protein conducting channel works and I don't want to go into a very great detail now let me quickly summarize a bit of work that we have done in E. coli and the E. coli translocation channel in the plasma membrane can be probably has a risen in evolution by imagination of the coli membrane of the coli plasma membrane to form the endoplasmic ellipsoid in fact the proteins which form this channel namely the so-called second Y E and G proteins where the first channel proteins were discovered in John Beckman's lab by Thomson Harvey and also by Newton Japan so they were really the first channel proteins which were discovered second Y E and G because it wasn't known at this point that they were channel proteins it was just known that temperature-sensitive newtons in these proteins inhibit translocation across the bacterial plasma membrane and so in evolution you can think about the plasma membrane having invaginated and then form the endoplasmic ellipsoid so that that channel which translocates proteins across the bacterial plasma membrane is perhaps fairly similar to the channel in the ER in fact the signal sequence which opens this channel in bacteria is very similar to signal sequences of secretory proteins in the endoplasmic reticulum and so this allows essentially to express insulin or pre-pro insulin in bacteria and get secretion of pre-pro insulin across the plasma membrane and this is important for genetic engineering and for becoming the production of this protein and the signal peptidase of E. coli will then leave the signal peptide at the correct side and you get secretion of co-insulin into the periplasmic space but anyway, so we were looking at whether we can detect a channel also here in the procularly plasma membrane so you can break up the bacteria into vesicles either inside out vesicles where the signal peptide binding site which faces usually the cytoplasm would now be exposed to the outside of the vesicle and you can also have right side out vesicle right side vesicle where this side is on the outside but we made inverted vesicles and then used the planar layer trick to fuse it to the lipid bilayer in these two chamber systems so this is the chambers again the trans chamber, the cis chamber we added the vesicle so if you add the vesicle, the inverted vesicle the signal peptide binding site should now be exposed to the cis side and not to the trans so what we then did is we made a synthetic signal peptide we have postulated in 1975 that the signal peptide would function in gating the channel so very much it would very much be like a ligand gating channel as you know the aspect column is for instance an example of a ligand gating channel where aspect column the ligand opens the channel and here we postulated that the signal peptide may have that function and may open the channel we synthesized a signal peptide by a chemistry mechanism and we added it to this cis chamber in the hope that it would open the channel now when we did this we got a tremendously noisy record because we did it in physiological soil concentration and what happens in physiological soil concentration the signal peptide binds and then the channel opens but it comes off again in the closest tremendously noisy record it was very disappointing it was almost that we struggled with it and we didn't know what to do with it and then by accident suddenly Simon one day added the signal peptide at high salt at 500 millimolar salt now remember the signal peptide is hydrophobic so in high salt you reinforce hydrophobic interactions so in high salt the signal peptide will remain bound to the signal peptide binding side of this channel is full there is something coming along nothing is actually coming other than the signal peptide and it opens and that you will see in the next recording which I show you when you open when you add this signal peptide you can see these very nice increases again in conductance this is done in high salt in each of these conductance step at 50 millimolar salt and you recalculate this for 50 millimolar salt all of it was done in 500 millimolar salt but when you calculate for 50 millimolar salt it's 220 picosemals so it is the same conductance properties that we get with the signal peptide and opening the channel in the traceminal membrane of the E. coli then that we get when we purge, when we clear the coating conduction channel of the endoplasmic epiculum by pure mice so the conductance properties of both channels are the same and of course when I mention you add high salt the signal peptide remains bound to the channel and the channels don't close they always remain open for conducting these ions and of course if you lower the salt concentration they all close because they come they get the noisy record again you get the very noisy record again so it's very clear that the signal peptide can be a ligand whether it is the only ligand for opening the channel of course there's another question in the case of co-translational translocation in the case of the ER it is very clear that the virus zone plays some role in keeping at least the channel in a stabilized configuration whether it actually plays a role in opening the channel there's another question and remains to be addressed now of course since we have SAC61 we can now reconstitute the channel proteins and can see whether we get the same conductance properties that we have observed with the native membranes and so far we haven't been successful in doing so but maybe because we need the SAC61 we need other coordinates to do this sort of thing for instance PIP or other proteins so we have so far not been able to use this approach because reconstituted polyoliprosomes to get these sort of recordings we think that the reason for that is because we don't have all the components that are present in the native membrane okay so this is okay this is just another control if you add the signal peptide to the periplasmic side you don't get anything then if you add it to the cytosonic side you get again here's two steps one step another step opening of these channels so this is the control that is really inside specific phenomena now so in summary then what we know is that I have three more slides which I will talk about in a little bit in summary of this where so the signal peptide in high salt can bind to these both inflammatory channels and can then open a channel and here's the corresponding recording you can see one single step increase in conductance and now if you go and clear the chain you can see again this increase in conductance so we have cleared out one channel and now if we remove the ribosome we increase the salt concentration we can remove the ribosome and then the channel closes you can see the record here and you can see here so this of course in summary of course is a very highly unphysiological maneuvers that we have to use to reveal this protein conductance channel and this is the reason why it was so difficult to demonstrate and this is why it was so much fun to chase it because it was really sort of totally unknown territory nobody had tried to do electrophysiology on a protein conductance channel and it is also clear why our friends who tried patched channel didn't know anywhere because you know these channels are either closed or when they have the process of conducting the chain they don't conduct ions and they are electrically silent and it was a great deal of fun to actually do these experiments and to learn something about electrophysiology now in the last couple of minutes I just wanted to demonstrate to you that nature has invented this protein conducting channel many times over one would imagine that once it has invented it, once it would use it for all the other systems for all the oxygen and so on but the great surprise was when we looked at the chloroplast out of membrane that in fact the channel there looks completely different and works by a completely different principle whereas this ER channel SAC61 is a highly hydrophobic protein which probably has some antiphilic helices which then because we know from Tom Rubberport's work Chris Ackies' work that SAC61 forms a tetramer or pentamen and there must be some antiphilic helices which line the aqueous core of this channel in the case of the chloroplast it appears that the channel is made up of a molecule which looks very much like a pore so it is not hydrophobic at all in fact it doesn't have a single hydrophobic alpha helix spanning the membrane it all appears to be better through the cheats than any one of the paradigms a channel with a positive literature is something like each protein has something like 26 better through the cheats crossing the membrane now let me show you how we got the channel in the chloroplast membrane and I think this is work of Danny Schnell in our lab and Felix Kassner and this is again I think very nice work because it's a very nice method and this here is the chloroplast membrane and I'm already summarizing what they found there are so called YAPS which means translocation intermediate associated proteins actually a terrible name and these are the molecular weight there is YAPS86 YAPS34 and YAPS75 and there is also a unique heat shock protein which is associated with this channel and so what Danny Schnell and Felix Kassner did is they took a precursor which had a signal sequence associated with it and as you know in chloroplast there have to be two signal sequences one which opens the channel in the outer membrane and another one which opens the channel in the inner membrane so what they did is they did they took this precursor which was expressed in the coli and then added urea to it to unfold it and then diluted out the urea precursor into chloroplast, added ATP and GTP and known for how you get import into the chloroplast but if you do a time course you get import in stages you first engage the channel in the outer membrane and then you engage the channel in the inner membrane and what Danny Schnell used they put at the carboxy terminal of this precursor protein a staff A protein presumably would be folded and would not allow the complete transfer of the protein into the lumen into the stroma of the chloroplast and would eventually yes because there are H2O proteins and so on and so on but it would slow it down a bit and so we were hoping that we would end up with a stuck translocation intermediate which would then after detergent solubilization using the staff A protein which sits out here be able to pull out not only this stuck translocation intermediate but with it the channel proteins and lo and behold this worked at least for the outer membrane components of the channel and I show you this very briefly here this is incidentally a very nice electron micrograph we are trying to focus this a little bit it's better than that where we took the chloroplast put it on a grid stained it very lightly and then used IgG gold particles to label the staff A which hangs out into the cytoplasm and you can see that the gold particles do not label the surface of the chloroplast randomly but label them in these sort of ridges and it had been earlier shown by staining color rather long time ago the freeze fraction of the chloroplast gives you ridges of particles in the outer chloroplast and in the chloroplast membranes so what we think is that these distribution of nonrandom distributions of these translocation intermediates on the surface of the chloroplast represent the nonrandom distribution of these protein conducting channels in the chloroplast membrane so I think this is all maybe I have one more slide no here is actually the data slide for that so if we this is my last slide if you do the incubation this is precursors for a very short time and then pull out these are the total chloroplast membrane proteins you see that you pull out a subset of proteins this one, this one and this one this is the precursor here and a bit of the process from the precursor that we used and this is a done in service name you can see that you pull out nearly stoichiometric amounts of this this band actually turned out to be two bands because there is a bit of HSP 70 by protein where it is now if you do the incubation a bit longer for 2.5 minutes you also engage the channel in the inner membrane and you pull out two more proteins this one and this one in addition now we have cloned and sequenced all of these proteins these three, four proteins actually only three we haven't done the HSP 70 type because it presented some difficulties and what we found is that I am really going back here to the slide to explain you in summary what we really found sorry this is the end of my talking that these two proteins YAP86 and YAP34 are two GTPases so it is very much like in the case of the endoplasmic eticulum that you have GTPases which sit in the membrane and we assume that there is under physiological conditions some signal recognition factor which recognizes the signal sequence and then targets this entire complex to these GTP binding proteins like the YAP86 and YAP34 very much like in the case of the ER where you have the SOP receptor after and better substance and so then you have this YAP75 which I told you looks like a pore like a porn it has not a single after a helix spanning the membrane but it has only these better few cheats and then there is in sub-stopmetric amounts like protein which may play a role in preventing backward fluctuation of this protein across the membrane and that may help in the transportation process so in summary then let me turn on the light again here in summary then I have told you a little bit about protein transportation across these protein conductive channels in the ER and we have now isolated in the case of the ER or the candidates for the protein for conducting channels in the ER have been defined and so also they have been defined in the case of the chloroplast of the membrane these also are the only membranes for which channel candidates have been identified in mitochondria they have not yet been identified and neither have they been identified in the oxysomes neither have they been identified in the thylacide membranes and so on and so on so we have a long way to go to understand how these channels work and this work is now past the inventory stage the inventory stage is over in this field and it's now going to move into biophysics and into structural biology and so this will be the next challenges to understand how on the biophysical level these channels work and how they do their work incidentally in the ER you have about in the average cell about 1 million channels of protein conducting channels and in the chloroplast we have estimated you have about 3,000 protein conducting channels in the other membrane tomorrow I will talk about the channel which constitutes the nuclear pore complex or which is in the center of the nuclear pore complex has a diameter 10 times larger than the channel in the endoclasmic reticulum which is estimated to be 20 angstrom in diameter and the channel in the center channel in the nuclear pore complex has a diameter of 250 angstrom so in this case of translocation across the unidirectional translocation systems you need to have the protein unfolded protein translocation can occur only in unfolded configuration and so if you have heat shock proteins helping you to keep the protein in unfolded configuration in the case of transport across the nuclear pore complex because the channel is 250 angstrom in diameter heat shock proteins do not play a major role in translocation it's not occurring unfolded substrates but I will talk about this much more tomorrow thank you very much there are any questions so obviously you can open the channel by knocking out the nascent peptide sequence with forum icing on the other hand presumably the ribosome is still bound so it does not occlude the form on the other hand you can open the channel by adding the signal peptide which may bind to a certain receptor side and not occludes the form so do you know what occludes the form is it an additional sequence it's a nascent peptide there is a question for the audience for the question the question was whether when you add the signal peptide and you keep the channel in an open front iteration at high sound and nothing else what would occlude it would the nascent chain occlude it the answer is that's what you think also we haven't found the experiment do you have further comments ok the experiment hasn't been done how do these channels compare with the channels which are formed with something so diverse as polysyns on one hand on the other hand the crystal protein of the basilar genesis which seems to be a very nice strategy to put both and kill the cell in this way so that it spills all the liquid nothing has changed he always gets in the first row and he asks very precise questions and he speaks very loudly so that you probably all have further questions but I will defeat it nevertheless he said what is the difference between these protein production channels that I have just described and let's say something like polysyn or let's say pterotoxin which can form the channels now I would call these private transport systems taxis of biology because the pterotoxin betters up and informs the channel so that it can transport the ultrasound and the same is true for some of the other systems like rice so they are proteins which are designed to integrate itself themselves into the basilar and they are private transport systems those are public transport systems where you have a channel which is designed to take a very large amount of whole spectrum of secretory proteins which go from very highly charged to very small to also highly charged and all what you need is a ticket a signal sequence which is a library to open this channel so this channel is constructed to provide access for a very large number of proteins to go across whereas the other channels are private transport systems, taxis which are designed for very few molecules where you can trick actually the pterotoxin it can conduct also as far as I remember the alpha subunit of ricin if you make a disulfide bond between the better subunit and ricin and the pterotoxin alpha subunit you can trick them too and there are probably many other channel systems which I haven't described in bacteria which are very fascinating for phage exit F1 for instance and this channel has to go through a channel in the outer minimum and so there are many other channels which we have to learn greatly about we know about proteins but we don't know how they function in terms of structure in terms of biophysics don't forget that not a single channel has been done by X-ray crystallography and so we don't know anything about the structure of channels this will be for the next generation hopefully we will be able to do the X-ray crystallography and we will have the 2.5 to 2-axis resolution of all of those channels and then we will know exactly that we may know any other questions? if not I would like to call your attention to the fact that there is a lunch for graduating students in 6630 biochemistry and graduate students will meet with Dr. global tomorrow at the 1230 and that the lecture tomorrow is in the same place at the same time 330