Protein Localization Inside Cells: Historical Experiments and Signal Hypothesis Explained

[00:00:12]hello my name is Manu heg I'm a group leader at the MRC laboratory of molecular biology in Cambridge England and in the series of three talks I'm going to talk about how proteins inside of cells get compartmentalized among the different organel now since the 1950s it's been possible to prepare biological specimens so that you can examine them by electron microscopy and this allowed people to see with unprecedented detail all the intracellular compartments of the cell and so if you look at the picture behind me this is a picture of a liver cell and what you can see is that there are numerous compartments and I've marked some of them here in different colors so there's the endoplasmic reticulum over here in green mitochondria in red and peroxisomes in blue in the nucleus near the bottom and the advantage of having all these different compartments is that you can specialize biochemical reactions in different parts of the cell without interference from each other now the consequence is that you need to put specific sets of proteins to carry out those biochemical reactions in each of the different compartments and so that raises a problem for the cell all of the proteins that are synthesized with very few exceptions are synthesized in the cytool and so in this picture you can see there are lots of little black dots and these are ribosomes and so in an average cell there are many millions of ribosomes and each of these ribosomes especially in a metabolically active cell is synthesizing approximately one to two proteins every minute so that means that every single one of these millions of proteins every minute has to be segregated among all these different compartments with High Fidelity in order to be able to make each compartment unique with respect to the proteins they have and so you have to take these to the endoplasmic reticulum mitochondria peroxisomes and many of them of course have to stay in the cytool and so how this is actually accomplished has been one of the major cell biological questions for over 50 years and so what I want to talk to you today about in the first lecture is a lot of background in terms of what is known about how proteins are actually selected and taken to specific compartments and what I want to tell you about is the historical experiments that led to the basic framework for how this happens then in the second and third lectures I'm going to get to much more modern experiments and in one of the lectures I'm going to tell you how cells deal with errors in this process because it turns out that it's not possible to get every protein to the right place with High Fidelity every time and the cell has mechanisms to deal with errors in these processes in the third part I'm going to tell you about how the cell actually maximizes Fidelity of recognition so that you can take proteins to the right place so let's get started now the simplest way to uh think about compartments is probably the inside versus outside so all cells have an inside and an outside and that means that all cells have to compartmentalize proteins so if you look at bacteria for example they're relatively simple and they have to secrete proteins from the inside to the outside for example digestive enzymes are secreted um as well as mating factors and other types of uh proteins the same thing goes with yeast as well as of course us so the cells in our body secr various hormones and while some of these hormones are small molecules many of them are proteins and these proteins somehow to get from the inside of a cell to the outside of the cell and so our understanding of how proteins get compartmentalized in cells actually comes initially from trying to understand how cells secrete proteins and how they actually move across the inside of a cell across a membrane barrier to the outside so the problem becomes apparent if you think of the scale and and scope of the problem so if you look at a couple of proteins here depicted uh here there are two different proteins that are depicted and then there's a cell membrane that separates the inside from the outside of the cell the question is how do these big proteins get across what looks like an impenetrable barrier and there's two problems with this first of all you don't want to secrete every protein so you have to somehow get selectivity so that you secrete one protein here but not the one over there and how does that accom how is that accomplished the other aspect of selectivity that has to be solved is you need to secrete these very large proteins and yet somehow not leak all of these contents that are inside the cell that are much much smaller so for example the cell may have various sources of energy like ATP or sugars that you don't want to leak outside the cell and so how do you get proteins across a membrane barrier without leaking all of the contents at the same time now the problem for bacteria is to just as I depicted in the previous slide move something from the inside to the outside but in ukar that is uh cells like us or yeasts the problem is a bit more complex and you don't actually secrete proteins directly across the cell surface instead a clue to how proteins get secreted in these ukar came from examining different types of cells so when you look at cells that are secreting lots of protein for example this anbody secreting cell over here in the right uh on on the left sorry uh compared to a uh cell that doesn't secrete many proteins such as this T lymphocyte you can see that they differ in their morphology and one of the Striking differences is that the cell on the left contains all these intracellular structures which compose the endoplasmic reticulum and so there was a strong correlation between cells that have to secrete lots and lots of things for example the cells in your pancreas or your liver versus cells that don't have to secrete very much and the abundance of the endoplasmic reticulum and so there was this idea then that the endoplasmic reticulum might be involved in the secretion of proteins and a clue to this came if you radioactively label cells with a short pulse of radioactive amino acid and then you ask where does that radioactive amino acid show up in the cell and so there are methods to look at uh cells in considerable detail for example you can look at this ER here and if you pulse this cell with radioactivity where you it first showing up is in fact in the endoplasmic reticulum and so it shows up here in the ER and then at slightly shorter times it shows up in vesicles that are distributed near the ER and then at even later times it shows up in compartments that turn out to be the Gogi apparatus and eventually the radioactivity winds up outside the cell and so the question is how does it get among these different compartments and it's clear that there's some type of secretory pathway that transits through the cell across these different compartments eventually to the outside of the cell and this broke the problem down into two subsets the first part of the problem is to get a protein into the endoplasmic reticulum so here you have a ribosome that's sitting on the surface of the ER and that Protein that's being synthesized has to go across the membrane into the ER which is green here now after you solve that problem those proteins then are carried by vesicle which is shown in yellow over here and eventually out to the surface of the cell so if you want to understand secretion you have to understand both parts of the problem and the part that I'm going to talk about is how proteins initially cross a membrane barrier here at the endoplasmic reticulum so the question then is how do proteins get from the cytool which is here the compartment in Gray here into the ER and there were a number of different things that were known uh for some time already for example ribosomes that are synthesizing proteins that are in for the surface seem to be Co on the surface of the ER so that already tells you that you're making the proteins in close proximity to the compartment where you want to send that protein the other thing that was that gave a bit of a clue is that if you look at the RNA that codes for these secretory proteins and translate that product the products seem to be slightly different in size in fact slightly bigger than the final product that gets secreted and that suggested that maybe it had some type of extra bit that told it where to go but really an understanding of how this process actually worked and how you got selective protein import into an organel like the ER wasn't really understood until you started to take the pieces apart and so the idea is that could you take a process such as this and break it down into different compart uh components and get those reactions or at least part of those reactions to work inside of a test tube because if you could then you could manipulate those components to try to understand the sequence of events that lead a protein from the cytool into the endoplasmic reticulum so this is exactly what was done by uh Bernard doberstein and gter blobel in in the 1970s and what they did was they isolated the different components needed for this process so there are three main parts so first of all of course you have to have something that will synthesize your protein so you need a translation extract and that typically contains a source of cytool and in this case the cytool came from reticul sites and this was chosen because reticulocytes are in the business of making lots and lots of protein in particular hemoglobin and so this was a great source of all the factors needed to synthesize proteins then you need a way to track the protein that you're synthesizing and that was served by using a radioactive amino acid and the most commonly used one is S35 labeled methine that is sulfur in the methine is labeled with a radioactive isotope of s of sulfur in addition you typically add a number of other reagents for example energy other amino acids trnas and so forth and all the components needed to get translation to work now the second part of the equation here is you need the ER membranes itself so what you can do is take ER from let's say a cell or a tissue that has lots of it and the classic source of this is the pancreas and then you can break open those cells and then use centrifugation that is spinning the sample to isolate components that are different in uh their uh properties so for example their overall size or their density and so in that way you can isolate rough ER and it's called rough because the ER studded with ribosomes on the surface now in order to not contaminate uh the ribosomes that are coming from the ER with ribosomes that are in the translation extract you can also strip these membranes and therefore get a source that has just the ER membrane and its contents now the third part that you need to try to get this reaction to work is a source of mRNA that codes for a secreted protein now you have to remember that this is before recombinant DNA technology was developed and so the only sources of mRNA was to isolate it from a native source and so what people did was they looked at cells that are secreting lots and lots of very small numbers of proteins so for example this antibody secreting tumor cell is secreting a huge amount of antibody and that antibody is composed of imunoglobulin heavy chain and imunoglobulin light chain and so if you take total RNA from this Source it will actually compos be composed of primarily two different RNA those for heavy and light chain and so you can then take these three components the pancrea uh the pancreas derived ER the imunoglobulin MRNA and the translation extract and mix them together in different combinations to try to see what is required and how this process actually works of getting a protein that's synthesized here in this this translation extract to eventually be inside the Lumen of the ER vesicles and so here I'm just schematically depicting the kind of results that uh Lobel and doberstein saw and so what they did is if they synthesized the MRNA for a secreted protein for example the imunoglobulin and they did this in a reaction without ER which is shown on the left here um you see one primary translation product and the way this is being detected is after you do the translation you can separate all the proteins by size on a gel and then you can detect just the newly synthesized protein by exposing that gel to film and detecting where the radioactive products are because remember even though the extracts have lots of proteins in them only the newly synthesized ones made from the RNA that you've added will become radioactive now something different happens when you do the reaction in the presence of these stripped ER vesicles and what happens there is you get primarily product that's slightly smaller and so in the this method of Separation smaller things migrate towards the bottom and larger things towards the top and you can see that there's a small change in size here and so what what is what exactly has happened here and so one clue to this came if you take these reactions after you synthesize the protein and add protease proteases of course digest proteins and what you can see is this smaller product is completely protected from proteas whereas the larger product on the left is digested to these small fragments and so what they deduced here was that the small fragment might have gotten inside the Lumen of the ER vesicles and that's what's shielding it from protease and they could demonstrate that because if you disrupt that membrane of those vesicles with a little bit of detergent then the products get completely degraded now you get a completely different result if you translate a cytool mRNA for example you can translate hemoglobin mRNA and there you see no effect seen when you translate with or without the ER vesicles and in both cases the proteins are completely degraded to fragments when you add protease so this along with a number of other pieces of information for example the fact that it takes time for this larger product which they suspected was a precursor to get converted into the smaller product over here LED them to propose a model for how a protein might get imported from the cytool into the ER Lumen so here's a picture of what they've what they imagined was happening and this is a figure taken from their 1975 paper and this overall idea is referred to as the signal hypothesis so the so of course this diagram is not particularly well labeled and so to help you out to see what they were thinking let's zoom in on parts of it and I'll label what the different pieces are so what you can see here is that what they've depicted is the MRNA in red and I've depicted the ribosome here in uh blue for the large subunit and yellow for the small subunit and Aug is the codon for start codon so what they imagined was happening was that you would start over here and the ribosome would start and the first little bit that it synthesized which they've depicted as a squiggly line they imagined coded for what they called a signal sequence or a signal peptide and so that little bit would get synthesized which I've highlighted here in dark blue and that sequence would not eventually be part of the full protein and the idea was that that sequence had the property of being able to engage with some Machinery that's in the surface of the endoplasmic reticulum and drive the import of that protein across the membrane now that little peptide that signal sequence would get removed by some enzyme in the ER so that then at later times you would continue to synthesize the protein until you were left here with a completed protein and by that time the signal peptide would have been removed and so that way when you look at the final protein it looks like the exact same size as the authentic product that gets secreted and so you don't detect the the fact that it used to have a signal peptide for a short time during its synthesis and then eventually the ribosome finishes translation gets recycled into subunits and then it can go on to translate more proteins and so the beauty of this idea is that it demonstrated that perhaps there were specific sequences that informed the uh protein where to go in the cell and so for the case of the ER those sequences would tell it to go to the ER because it would have way of being recognized by components perhaps in the endoplasmic reticulum itself so that difference of having or not having a signal peptide is depicted as this change in size and they imagine that this represented an extension at the end Terminus of the protein so what exactly is the basis of this extra mass and so at the time it was very difficult to identify the sequence of that directly in fact the amounts of this protein that you're making is extremely small although it's radioactive so you can detect it other ways of detecting what's going on were very difficult so they had to use a clever trick to figure out what the sequence of this extra little bit of sequ extra little bit of protein might represent so remember that uh these translations are done in the presence of radioactive amino acid so let's say that up here this is the actual sequence that's in your protein of course you have no way of knowing that and so how can you figure this out and the way they did this this was they did the translation reaction in the presence of radioactive uh amino acids and different amino acids in different reactions so in the first reaction they use radioactive seene for example and so what you'll see is that a Serene gets Incorporated wherever there's a codon for that amino acid in this case the sixth position the 11th position and so on so how does that help you well it turns out that you can take a protein and sequentially remove amino acids from the end terminus and you can see if that amino acid that you've removed is radioactive or not and you can do this by counting the number of radioactive counts in that sample so suppose you do that and this is a process called Edmund degradation because you degrade the protein but one amino acid at a time and what you would see is you would see a spike of radioactivity when you get to the sixth amino acid that codes for Serene because that's the one that's radioactive and you'd see another Spike of radioactivity when you get to the 11th one and so on and what you can then do is repeat the whole experiment but with a different radioactive amino acid so let's say Proline or Lucine and that would tell you the positions of each of those amino acids and then you could piece this together and of course you wouldn't know the whole sequence but you would know for example that there were a bunch of Lucin here there were perhaps two lysines in the sequence and so forth and when they did this with a number of different signal peptides they noticed some patterns that emerged for example a lot of signals had stretches of amino acids that were hydrophobic so in this case Lucine which is a very commonly found signal commonly found amino acid in signals they also noticed that signals were roughly 15 to 30 amino acids long and occasionally they seem to be enriched in basic amino acids near the end Terminus and so this provided a overall idea of roughly what signal peptides look like and initially although it was thought that they might have some specific sequence homology in turns out that they only have these General features in common and in the third talk I'll discuss a little bit about how despite these very generic features they're nevertheless recognized efficiently so now that one has information about what the sequences are that perhaps direct a protein across the ER one wanted to Now understand what is the Machinery that's recognizing these signal sequences and directing them to the right part of the cell and so there were two strategies that were taken one was a genetic strategy which was uh to exploit microbial systems such as bacteria and later yeast to try to identify mutants that were defective in secretion and this was an ERA when recombinant DNA technology was just emerging and so what one could do is to take the signal peptide of a protein of interest and fuse it to a reporter that you could easily assay if it got secreted or not and so then you would look for mutants of either bacteria or Yeast that were defective in translocating the Pro prot protein across the membrane now at the time it was not really clear whether the process of secreting proteins in bacteria for example was related in any way to getting a protein into the endoplasmic reticulum of eukaryotic cells so in the mimion system for example a different approach was taken because genetic strategies were would have been much difficult at that time and here the strategy was a biochemical fractionation strategy so this is analogous in many ways to the biochemical Strate that was initially used to decipher the signal hypothesis which is that you take different components of this biochemical lysate that reconstitutes a process of interest and you fractionate it and dissect it to try to identify not only the sequence of events but what factors are involved in a process that you're interested in so here's an example so let's say you have native ER microsomes and so this is something that has all the different proteins that are in the ER and at least some of those are involved in the process of getting a protein across the membrane into the Lumen of that of that compartment now if you want to try to find some of those components one thing that you can try to do is to somehow separate the components and so one strategy for example is to use high salt and if you treat these membranes with high salt the proteins that are associated peripherally on the surface wind up getting liberated which is shown on the left and proteins that are either inside or in integral to the membrane remain with the vesicle and what you hope is that while the initial starting material was active for translocation of proteins across that membrane that the other components were inactive now of course it's very easy to inactivate something biological the real trick here was to hope that the components that you had extracted when you add it back restores the activity to this inactive membrane and so what that tells you then is that something that you extracted off the surface with high salt must be important for the process of translocation and so you could then take this material here and you can see that there's a a limited subset of components there and then fractionate that further let's say separate it by size or charge or other biochemical properties and then you check each of those fractions for the ability to complement the activity of what was otherwise an inactive membrane fraction and so exactly this was done by Peter Walter when he was in Gunter bobal lab as a student and here's a picture from the paper that described the purification of such a factor and so what you can see on the top is a number of different fractions in this case uh Separation by size and some of those fractions if you check them for a translocation activity convert the precursor of a secreted protein into a processed form and that remember that that shift in size is indicative ative that the signal peptide of that precursor is being removed which is an indicator that it's successfully getting into the ER vesicles in this reaction and so if you look at those same fractions for what proteins they have they they it look like they had six different proteins now the ones here near the bottom are very hard to see because they're quite small but you can see that the fractions that contain the most amount of these proteins also have the best activity and so this suggested these proteins which couldn't be separated any further somehow form a complex that is responsible for part of the process of getting a protein into the ER and so this complex was initially termed signal recognition protein but it turned out later that actually in this complex was a large RNA and that was actually discovered accidentally because when Peter went to go measure the concentration of his protein you can do that by measuring the absorbance at uh 280 nanm somebody had set the spectrophotometer at 260 nanm which is optimal for detecting nucleic acid and when he got a really high reading he recognized that there must be some nucleic acid in the sample and rather than ignoring that result he recognized that there was some RNA here that must be part of the complex and so this is now called signal recognition particle not signal recognition protein so he did a whole series of experiments because now he had purified SRP which is signal recognition particle and he also had the ability to synthesize cytool proteins so he could have cytool uh protein synthesizing ribosomes and ribosomes that were in the process of synthesizing secretory proteins and he could then test what the properties of SRP was relative to its ability to engage with either of these two types of ribosomes and what he was able to show is that Sr binds very strongly to ribosomes that are in the process of synthesizing a secretory protein but binds rather weakly to a ribosome that synthesizing azolic protein so the idea was that perhaps SRP is weakly binding and coming off of all ribosomes but once it recognizes one that has a signal peptide that's uh exposed from it it would bind much more strongly to that in addition SRP could serve as a mediator that would allow ribosome syn sizing secretory proteins to bind to the ER membrane and that kind of binding activity was not observed with ribosome synthesizing cytosolic proteins so that was another difference and that also suggested that SRP was somehow important for targeting these ribosomes to the membrane and in fact in subsequent studies using SRP as an affinity handle they identified the receptor that it binds to at the surface and so SRP he then takes ribosomes that are exposing a signal peptide to a receptor and that receptor is located at the ER membrane the third observation is that when they added a high amount of SRP it would actually arrest translation of proteins that were synthesizing of uh ribosomes that were synthesizing secretory proteins now it didn't have such an effect on cytosolic ribosomes but it turns out that this arrest while concept very useful because it suggested that these proteins that were synthesizing secretory proteins these ribosome synthesizing secretory proteins would somehow stop translating until they got to the membrane turns out probably not to be correct because in systems that are homologous that is where all the components come from the exact same system rather than the systems that were used here where components come from different sources this arrest is very weak or not observable but but it was a conceptually interesting idea at the time and it basically suggested a sequence of events in which if you have ribosome synthesizing either secretory or cytosolic proteins SRP would selectively recognize the signal peptides that are on the secretory protein then it would have a receptor that was at the ER membrane over here and somehow that protein would get transferred to what was hypothesized to be a translocation Channel or a translocon and then with subsequent synthesis the protein would go across the membrane through a hole in the membrane formed by this translocon and at some point the signal peptide would get cleaved and eventually it would get translocated across the membrane of course the cytosolic protein would complete synthesis in the cytool and stay there and so this was a scheme for explaining how you could get selectively some but not other proteins recognized targeted and translocated across a membrane so a big question now of course is what exactly is this translocon and in fact it was debated for many years whether you actually needed something and this was because the signal peptides were known to be hydrophobic and because the interior of the membrane is hydrophobic many people thought that perhaps the hydrophobic signal just inserted spontaneously into the hydrophobic membrane Bayer and that somehow triggered transl ation but um others thought that that was very unlikely and that there should be some type of pour through the membrane formed by proteins and a search was on for trying to identify what this translocon was now there were a number of different problems with finding this first of all the translocon would be a protein that's membrane embedded and those tend to be much harder to isolate and purify the second problem is that there was no obvious assayable activity it's not like it was an enzyme for example the enzyme that cleave signal peptides which turned out to be much easier to purify and third it's only transiently expose uh engaged so it's used but then it's no longer engaged anymore and so how do you go about finding what these uh translocon protein or proteins are so an important Advance here came when it was discovered that it was possible to stop translation precisely at specific points during synthesis of a protein and so this and uh took advantage of recombinant DNA technology so suppose you have a plasmid that has a promoter and encodes a secretory protein of interest and of course that's going to have certain restriction sites within it so for example I've labeled e R1 here in the middle now if you cut this plasmid with Eco R1 and then you transcribe that cut plasmid what you'll generate is an mRNA that is a fragment of the whole mRNA that codes for the protein so it will only be the part that codes up to the Eco R1 site if you now put that mRNA into a translation system the ribosome in that system will translate to the end and it turns out stops at the end of that mRNA so that means it will have synthesized up to the part that encodes uh the protein to that point in in the sequence and so of course if you cut at a slightly later point for example at a bamh1 one site and then translate that you'll generate a longer product and so what that means is you can take a continuous process such as this that usually occurs co-translationally and actually discreetly break it down into distinct steps because you could stop translation at a very precise point and so this then could be exploited for example to see what was happening at early versus late steps and once you could generate such intermediate it was then possible to see what proteins were around your synthesizing protein the protein you're you're trying to synthesize so one way to see what what's around your uh nent Protein that's being synthesized is to use crosslinking cross-linking is a method there are many different versions of it but in short there are different methods in which you can covalently link two proteins that are next to each other so here's again a sample type of an experiment in which suppose you isolate a product that has synthesized up to the point where a signal peptide has come out of the ribosome so it can engage SRP and if you take that sample and treat it with cross Linker this is the kind of result you might expect now remember that the only things you can visualize here is the radioactive amino acids that are in your newly synthesized protein and so what you can see at the bottom is the non-cross Lin product so that's your your fragment of a protein that you've synthesized but if you add cross Linker that will at least some of it will shift in size and it will shift by the molecular weight of whatever it has become cross- link to and in this case it cross- links to a subunit of SRP called SRP 54 it's called 54 because it's 54 kilodaltons in size and it's these kind of experiments that provided evidence that this subunit of all the different subunits of SRP is the one that probably recognizes directly the signal peptide now if you make a longer intermediate and do the reaction in the presence of er membranes you might generate for example a intermediate that's in the process of translocating across the membrane and if you do the same cross linking the cross link to SRP disappears and now you see a cross link to a new component which one would hypothesize is a component of the transcation channel itself and there were a number controls that one would do to confirm this for example if you release your protein from the ribosome this cross link would go away suggesting that it was a component of the membrane that was only nearby when you were in the process of going across the membrane but not after and similarly you could confirm that the cross-link product was integral to the membrane that is it was a component that might for example have the property of forming a pore across that membrane and so so you could then use this as an assay to for example identify conditions where it was possible to solubilize the membrane but retain the interaction with the translocon and so it was determined that for example whatever the translocon was could retain its interaction with the with the ribosome even under conditions of very high salt in the presence of certain detergents and that allowed you to wash away all of the components that are in the ER membrane leaving behind just the most tightly Associated factors and then you could identify what those factors were and it turns out that when that was done from a Mamon system and small bits of sequence of that protein were determined that protein turns out to be homologus to two genes one which was isolated from yeast and another which was isolated from bacteria that had come up in genetic screens for defects in secretion and so that was then very satisfying because it suggested that a component that biochemically seems to be involved in the process of translocation is concordant with what's genetically identified as genes involved in secretion in microbial systems and that suggested that the process at least this pathway of getting proteins across a membrane is highly conserved across all organisms so the picture that emerges then is that you have SRP at the beginning that initially recognizes signal peptides takes the ribosomes to the membrane and then the protein then engages this this purple colored or magenta colored uh component which is the translocon and that then forms a conduit across which you translocate a protein across the membrane and this in fact is the picture that you now see in textbooks for how proteins are imported into the endoplasmic reticulum and as I mentioned based on both the genetics and the biochemistry it turns out that this pathway is universally conserved so all organisms cont contain some version of SRP SRP receptor which I've labeled Sr here and sex 61 not only that but it's the these factors are used not only for getting proteins across the membrane but also inserting many membrane proteins into the membrane and the way this is thought to work is that if during the process of translocation a segment of protein comes out that's hydrophobic that segment can laterally insert into the B layer forming a membrane protein and so you have a Machinery that's capable of moving proteins in two directions across the membrane and into the membrane and through a combination of these two activities you can imagine how this basic Machinery perhaps with the help of various other factors that remain to be identified might be able to make a wide range of both secreted as well as membrane proteins uh for the Cell at the ER so for example you might imagine that if a protein has multiple transmembrane segments it would just use these processes in succession to insert them in the membrane although many of these details remain to be determined similarly you could imagine that if a protein inserted but then it didn't get cleaved that those hydrophobic sequences could be again similarly recognized by SRP and sex 61 to be uh generating a membrane protein so then if we go back to this initial diagram where proteins that are synthesized in the cazol have to be segregated among all these compartments what are the general principles that we've learned from examining the process of getting to the ER so first it turns out that proteins have signals and this turns out to be true not only for the ER which is what I've depicted here but in fact you have different types of signals that take proteins to the mitochondri to the peroxisome or in plants to chloroplast and yet different types of signals that are used for import into the nucleus and so the general idea that newly synthesized proteins have specific sequences that are used as essentially zip codes to tell you where to take that protein um turns out to be very widely applicable the second thing is that you need recognition factors so in the examples that I've given you um the recognition factors for this pathway are SRP and sex 61 so SRP of course recognizes it in the cytool takes it to the membrane and then it turns out that the protein is recognized a second time by SE 61 not only to check that you've got the correct type of protein before translocating it but to also open that channel and third you need something that gives you spatial cues so in this case really the primary spatial CU is provided by the SRP receptor which sits in the ER membrane and so that way when you're recognized by SRP you take it to the corre direct compartment of the cell and finally you need transport factors of some type because it turns out that although there were many hypothesis about proteins being able to spontaneously move across a membrane using only biophysical means in all instances that have really been examined um you need some type of Machinery to form conduits across the membrane for translocation across the membrane or some way of moving that protein into the Bayer in the case of membrane proteins and so it turns out that these basic features are found in Transport Systems to the mitochondria and other organel and so the basic ideas developed from primarily biochemical analysis which then meshed very well with genetic studies has led to a framework in which one can understand how proteins get to different parts of this uh parts of the cell so of course there's a huge amount that remains to be uh discovered it turns out that there are still many Pathways that are still being discovered you might ask well why would that be and the reason is because in our bodies for example we have somewhere around 20 to 25,000 genes and they're incredibly diverse the proteins that are coded for by those genes and so it's unlikely that you can just use one pathway for each organel to get all the proteins that need to go to that organel to that spot correctly the proteins are simply too diverse and it's turning out that even as recently as just this past year or two there are new Pathways that are being discovered for how different types of proteins get to different organel the second thing is that the mechanism of actually moving the protein across the membrane remains unclear in many instances in the example I gave the protein is moved across the membrane while it's being synthesized which solves the problem of how you move a really big protein across a imper an otherwise impermeable barrier because you do it while the protein is still uh unfolded however there are systems for example certain systems of export out of bacteria or uh import of proteins into peroxisomes in which whole folded proteins are transported across the membrane and we have a very poor idea of how those kind of processes work then most of what we know comes from the study of very uh simple model proteins but if you want to understand how much more complex uh proteins particularly membrane proteins are folded and assembled um we have a poor understanding of that process and finally although these Machinery look like they're very sophisticated and it's amazing that the cell has evolved all of these Pathways to get proteins to different compartments it turns out that these Pathways do fail from time to time and so you need mechanisms to try to get as High Fidelity as possible and mechanisms for dealing with these failures which is a process called quality control and in the second and third parts of the talks I'm going to talk about both how quality control Pathways were discovered in in the context of protein localization and how the machinery for example SRP and sex 61 um achieve reasonably High Fidelity despite the fact that they have to transport lots and lots of different proteins through that same pathway so thank you for your attention and I hope you'll listen to the other two parts of this uh talk e