We are going to switch gears to talk about another independent line of evidence mainly lead by biochemist Jim Rothman, James Rothman. Originally at that time was in Sloan-Kettering, right now in Yale University. And he independently identified the key components for transmitter release. And again, I'm going to provide some background about his research and then it's your turn to design experiments to fuller understand our process, okay? So the title of his work is SNAP receptors implicated in vesicle targeting and fusion, okay? And look at the time, this is March 1993, okay? And the previous paper that we identify, this is 1992, okay? So the James Rothman's paper, again this is both of them are in nature. James Rothman's paper is just half a year later than this one, okay, in nature. Okay, let me just provide you a little bit background James Rothman, okay? Who recently got a Nobel Prize by his work, okay? And you can look at his sort of an educational background, essentially collecting all the good training in famous universities, right? Bachelor degrees in Yale, PhD in Harvard, post-art in MIT and then first become a faculty, assistant professor in Stanford. And then become a chairman at Stanford and then move to New York in the Sloan-Kettering. And then be in Colombia as a faculty and then before getting Nobel Prize, he moved back to Yale, okay. So he's basically in good places okay. And then this is before his Nobel Prize, he has been awarded many prizes. Okay, so next time you can be predicting how a person can win Nobel Prize by counting how many other awards they get. Okay, and the most important one would be the Lasker Award, right? Once a person get a Lasker Award, then more than about 50% of the time, he or she might get Nobel Prize, right? And this happened with some Chinese. Okay, so let's just discuss a little bit the approach he did and then you guys need to design for these parameters and how to interpret the results. So James Rothman, he's very interested about the again, the molecular mechanisms controls the vesicular fusion. Initially, no transmitter release, but a general principle controlling the membrane packet in a fusion. Okay, in all the cells, there is [INAUDIBLE] of the membrane protein from the circulatory pathway. From the [INAUDIBLE] to the plasma membrane. Okay he's very interested how those for example. Can only specifically deliver to the plasma membrane membrane to deliver some sort of membrane protein on surface rather than the will be infused with other like mitochondria, like [INAUDIBLE], like other organelles, you know? There must be some specific controls for those trafficking, if not then the cell will be in a bad condition. All of that component will be randomized. So he's trying to understand that. And so his previous work, and that is how the past that I'm going to provide you the background. So previously he's about chemist. Let's just take five minutes break, okay? Now, let's get back to the James Rothman's work, okay? James Rothman, he is a very smart and established biochemist. He's interesting used biochemical approaches to identify the key molecular components control the. In his previous studies, he already identified important factor called NSF, okay. That is very critical for membrane to fuse with past membrane, okay? How he do that? He's a biochemist, so he used ingredients to isolate the gorgi vesicle, and isolated component passed the membrane extract, okay. And here is the experiment, he designed an essay. He put enzymes inside the Golgi vacuoles. And pour substrate inside the plasma membrane components, okay? And only when these two fills, the enzyme will meet in the substrate and then he can see the reaction, okay? So in the essay he found that there is ATVs, NSF is record. How did he do that? Well, he found out using the Golgi and plasma membrane and if you simply put them together they will not fuse, but rather on top of this you add a cytoplasm extract, cytosol extract. Okay, basically, some components inside the cytoplasm. And with ATP and GTP, then these two will fuse, okay. As long as when this extract works. And then if this extract contains a protein, he can isolate the extract into different components. You can separate the extract by molecular weight. Okay, by the by the by the chemical sensitivity, okay so basically we can separate this extract into individual components. Eventually he found a very critical component, NSF, ATPS, but it was very critical for the [INAUDIBLE]. That's all his, more than 15 years work in five minutes, okay. He found the NSF, okay. But this NSF is an ATPase, okay. But it's a cytosolic protein, meaning that it doesn't not bind to membrane, okay. How do they work? How does it work? The problem is if you want to get membrane to fuse you only have a cytoplasmic protein. It needs to somehow attach tot he membrane, somehow handle the membrane to cause membrane to fuse, okay? So, he hypothesized there will be an important receptor for NSF, okay? That there will attach to a membrane, okay? So, this NSF is a cytocolic protein, you need to work on some membrane components to cause membrane fusion. That will make sense, okay? That's all he know and that's how he design experiment. Okay let's see how he do that and then you guys can help me to answer why he do that in this way. Okay so he identified NSF okay. So in his experiment he wants to identify what is the receptor in the plasma membrane? So how did they do it? Well first for the identification what they did was they first enrich the NSF-Myc, okay? So what is Myc? Myc is the is after that you confuse with NSF. And then you can recombinantly express this protein for example in coli. You get a lot of proteins, okay. And there you can get this commercial available link with the antibody, okay? So, once we use this piece, you can enrich NSF by to this piece, okay? And previously they already identified snap, this two proteins. Actually, they are the accessory protein or the binding protein for the cofactor for NSL that they already know, okay? For NSL to work, it requires the snap, okay? So then one idea is they are trying to use this. Please to put out NSF, this SNAP, okay? And then they want to add some print, some cell extract to put on the seeds, okay. Similar as some of the students say, I just need to okay? And then they want to identify which things they put up, okay? So, now here's the question. When they use this antibody, they add the and they add the. Why do they want to ATP gamma s and EDTA why do they want to do that? Okay, that's great so it's exactly like that. So NSF is ATPA we already talked about this, we already talked about we talk about kinase. Whenever you have an enzyme and how it works with suction. Usually its a reversible process, the request turnover right? So for example the proteins will cut the substrate and the substrate will get released, the LSF is ATPAs. Why it does just like any ATPAs, you will catalyze the hydrolysis of ATP and then undergo some confirmational changes to a fair substrate. Usually we'll break the substrates or make the substrate undergo some confirmational changes, okay? So if you are trying to identify the substrate for NSF, in this case the receptor for NSF. Then, In the presence of ATP we will always turn over okay? Has ATP, you will change the cell substrate, and a new cell substrate coming, and then you will get changed. So you will have a low affinity fighting to the substrates, okay? So what it did is using the ATP analog. ATP-gamma-S, okay? ATP has three phosphate, okay? The gamma is the part that one of the oxygens has been replaced with sulfate, S, okay? So it is the perfect analog for ATP, except this ATP comments because the S has been replaced, you know, with all the oxygen. It cannot follow be hydrolyzed, okay? It can feed it in to the ATP by the pocket to allow, to the, [INAUDIBLE] to adopt a confirmation to buy to the [INAUDIBLE]. It will not follow get hydrolysis to restop actual original state, so in essence, the ATP is a lock into an active confirmation just to grapple the substrate, okay? In a irreversible manner, okay? And the way they added the EDTA it's, I think, it's as somebody pointed out, let, this ADPA will [INAUDIBLE] magnesium. Okay, because when you synthesize ATP [INAUDIBLE]. Maybe there's some contaminant of ADP. Or maybe the cell, since you are going to get something extra from the cell. The cell might contain some ATP, okay? So, they added the EDPA to all the magnesium, and magnesium, ATP, they work together to work as a cofactor for the NSF, okay? So, in this case they can lock the LSF in this active confirmation and then they want to put out this component, okay? And how did it get this extract, okay? Get this extract, what are you going to do, where are you going to get this extract? Okay, great. First you need to get a lot of material for biochemical analysis you need to have a lot of material. The reason being, the proteins, they are not regenerative, they are not like PCI you can amplify the DNA. Well now we lose some proteins, they get lost, okay. And, secondly, you neet to retent the membrane fraction. How do you do it? Because that we, want to study the receptor, they caught, in the membrane that, been recognized by NSF. Okay so when they extract the component, they got to attend to the membrane faction. How do they do it? Well you can use some special detergent that can solubilize the membrane component to retain their confirmation okay? Detergent. That is hydrophobic and hydrophilic in a way that solublize the membrane and including protein components to keep their native structures, okay? This protein? Yes indeed you can get a cultured cell. But what it did interestingly that they get from a brain not why do they wash magnesium ATP gamma s and saying this is a non-specific elulate okay? Why do they want to do that? Okay so the reason that we are using ATP gamma S, because as we mentioned ATP gamma S is the analog of ATP. That it cannot be hydrolyzed in by ATPAs. Okay, so even if you add the magnesium ATP gamma S. We were trying to wishing with it, the sense that the key is loaded, would be the non specific sense of vice to it, because the ATV [INAUDIBLE] exist, you will still lock the NSF [INAUDIBLE] confirmation to buy to the specific packet, okay? Only the non specific packet which cannot be hydrolyzed. That it will get eluded. And then they will add excess amount of magnesium ATP. Because ATP and ATP compounds are analog are similar right? So ATP will compete out, its going to replace. And once the ATP replace the the ATP can be hydrolyzed by the ATPAs and then the NSF is going to undergo some conformational changes to release, to go down from an active state, to release the binding component, and this would be the specific.
