Okay, so they do that experiment. Again, in number one, it's the piece without an SF, okay? That is, you just add this piece, but you don't add an SF, okay? And then you go through this process, okay? Y of 10 is in this page [INAUDIBLE]. You see some components here. Okay, why now would there be some components there? What are they? Do you care? Why there would be no components, what do you think? They don't have a staff, why would there be some components there? Okay because what you have is even though there's no SF, you still have the beads. You still have this antibody there, okay? You might bind to something right? Hydrophobic interaction, non-specific interactions. And indeed, this is likely due to the bee's or the antibody coding how the bees can interact with some components. Okay, that's the reason they need to do this control. Because the purpose of this study is to identify the substrate for NSF, right? It's not to study the hollow piece, or the antibody itself can interact with some component. This is not the purpose of the study, right? And then, they wash, dilute, with magnesium ATP chrome s again. You can see that there's some. And this component is similar like number one, okay? And this is again because you are using ATP kinase and then you are this similarly like the piece. This is probably also nonspecific. And then this is the one that is go to mine. So they added a magnesium ATP and inculbation and low and behold YFI is aha! Now you've got something interesting. First, you've still got something here. Okay, I don't care about what's on the right because they're they're just similar like number one and number two, especially number two. We renounce specific binding. But in comparison between two and three. You see there's additional components A, B, C, D, E. Okay some of these things are a little bit weak so they label them. There's also F here, okay? So they can see in a much more convincing way here, okay? So the in comparison with number 2 they got more components here, okay? And this will be the specific ones because it doesn't exist in the ATP'S solution components, right, okay? And this is a goldmine, okay? ABCDEF. Okay, now what are you going to do? You're again, what if they already have these pure components? They're just sequence in this protein. They are pure, right? We have already done this three times. And that protein's experiment we identified a substrate, we sequenced it adamine degradation. We sequenced it from end terminal. We can identify which amino acid in the sequence right, and then we know which gene because of the central dogma. The gene determine RNA, and then we can translate it into the proteins right. You already count this a, b, c, d, e, f. You just cut them out and individually sequence them. That will be a 100,000 times easier than to try it, because you don't know what to try. Okay, so they sequence these components, okay? So they got A is syntax in B, and C is the syntax in A. Actually they are similar isoforms, okay? And then E is a and then F is one or two. Okay, bingo. If I am the author, I would be very excited to see those things. Especially F, are you excited to see this? F is labeling here. It's 1 over [INAUDIBLE] bribing. Two, I am very excited to see that. Are you excited to see this? The [INAUDIBLE] so if we fast backwards to this paper that we discussed Saturday, right? 1992, October 29. The group identify using this toxic, this tetanus and bot-B toxin. They said this toxin blocks transmitter release by proteolytic cleavage of synaptobrevin. Okay, they identified. This synaptobrevin. This synaptobrevin if you still remember. This is synaptobrevin is the key component that have been clear and after there's no transmitter release, right? In 1992 October, and James Rothman in 1993 after about half a year in the same journal Nature, using a totally different approach. They're trying to identify an ATPA that he started for many years. He wanted to understand these ETPAs, how you regulate vesicular fusion, okay? And then he identify this ETPA's specific target. At this one. It's also a. They are also using a extra. And previously some of the students using [INAUDIBLE] writing to do affinity purification to identify the binding partners, right? Probably the [INAUDIBLE] group just identified a [INAUDIBLE] in are happy in doing the experiments which they are proposing to do. So they are trying to prove that. And then the [INAUDIBLE] after half a year. Another group identified the contacts with the ATPA's packet. And there is additional, at least, two proteins, [INAUDIBLE] 25 and syntaxin will be in the contacts. And these whole contacts will be the packet for ATPase. So they might be happy or be crying, right? So they will be, happy is great. It's not working alone. As we predicted, it has some additional component, right? So this really makes sense, right? So for different approaches, why is coming from a toxic people identify the. And the other one who was trying to identify fusion, NSF especially, and then they also come up with the along with the other components. So in the early 1990's, the whole field are very excited, because from all the different approaches, people come to the cent components that might be essential for regulating transmitter release. Indeed, we don't have time to discuss, but Randy Schekman, who also get a Nobel Prize, through east, through the east genetics. Also coming up with the mutants that is homolock to the synatopribing, syanpt25, and the syntexted. Okay so in the early 1990s, so this is an east paper. Identify the complementation groups using yeast genetics important for membrane fusion. They also identify the whole mutant. So, in the early 1990s people are excited because of a converging evidence [INAUDIBLE] identify. [INAUDIBLE] actually located near the plasma membrane okay? Syntaxing, snap 25 are in the plasma membrane so why is it in the basal membrane and the other is in the plasma membrane. And they will follow context. Does that make sense? If you want to cause vesicle to fuse you will sort of predict something will go on the vesicle membrane, something will go on the plasma membrane. And somehow they work together to cause membrane fusion, right? So indeed, James Rothman after identify, the snap syntaxis and identify the synap worry, they use just this so called snare component. This is three proteins. They pool in the liver zone, they pool in the liver zone and then they demonstrate. In a liposome if you put them in the right amount, then they are sufficient to drive liposome fusion. So for example in this case this indicates that there's a liposome fusion, okay? So just putting this protein in different liposome itself It's sufficient to cause the membrane fusion okay? So all this evidence and if you are cleaning using a toxin then there's no fusion. Okay so all this evidence in vitro, in vivo evidence all together make the feel very excited. They identify the key components to cause membrane fusion and that is how snares was identified. And indeed from a structure people found actually this is similar, it formed this helical structure. And for virus to cause an infusion it turns out the virus using one protein, one of its three protein, the virus is interesting in a way. The virus will express a protein itself already has several helical domain. But the other side, once they reach the target it will insert into the target's membrane, and it will also undergo these similar structure arrangements. So now people get some basic ideas. There's key components there that might be important for Vesicle infusion. And then it turns out that the are not calcium sensitive yet, okay? And that's how it works, from and Alice identify a calcium binding, but also enrich in a synaptic vesicle a protein that is important for vesicle infusion. And that's not can physically interact with the so-called SNARE complex, okay? So therefore, all this is putting together, how different molecules located at different places associate with each other. And binds to calcium and then trigger transmitter reduce.
