BIO241 Lecture 6

Thursday, January 22, 2008 - Today we continue with sorting of proteins to the endoplasmic reticulum, another form of transmembrane transport. - Recall there are 2 types of ER: the rough ER (actively carrying out protein synthesis with ribosomes associated with it – the ribosomes are the things that give it the rough appearance) & smooth ER. We are going to focus on the rough ER today. - Proteins are sorted to the ER through transmembrane transport – recall nd st this is the 2 type of transport that we covered in the last lecture – the 1 type is gated-transport & then we covered transmembrane transport. Once proteins enter the ER from the cytosol & from there, they can be further sorted to other compartments such as the Golgi, lysosomes or even secreted to the cell exterior by vesicular transport. - Today we’re going to look at how proteins get from the cytosol into the ER by transmembrane transport. - ER is not a static structure, it is actually changing all the time in terms of its location in the cell & its composition in the cell. This video nicely shows that. - Video: This would be an ER network here & just take a look at how that network changes just as you’re looking at it over time. - Description: The ER is a highly dynamic network of interconnected tubules that span the cytosol of the eucaryotic cell, like a spider’s web. The network is continually reorganizing with some connections being broken while new ones are being formed. Motor proteins moving along microtubules can pull out sections of ER membranes to form extended tubules that then fuse to form the network. - This is just to demonstrate how dynamic this whole process – ER is very dynamic & proteins are coming into it at multiple locations where there is the rough ER with ribosomes associated with it.  Synthesis & modification of proteins (covered in this lecture)  Synthesis of lipids (covered in a later lecture)  Have ER signal sequence - The proteins that are sorted to the ER all have a signal sequence which is present on the protein so this would be the mRNA here (in the diagram), a ribosome associates with it & starts to translate that protein & the ER signal sequence comes out at the N-terminus – the 1 part of the protein that comes out of the ribosome so it’s an N-terminal endoplasmic reticulum signal sequence – ER signal sequence. - The types of proteins that have an ER signal sequence are both soluble proteins & transmembrane proteins & these are many proteins destined for the Golgi, secretion outside the cell & also to the lysosomes. Also some proteins are destined to stay in the ER.  Hydrophobic AAs at N-terminus - mRNA comes out of the nucleus & then ribosomes associate with the mRNA & to translate that mRNA into protein. Normally, what occurs is that multiple ribosomes will bind to an mRNA molecule & you’ll get polyribosomes in the cytosol associated with that mRNA & each one of these ribosomes can be translated into protein from this one mRNA molecule. That’s where all proteins start, most of them, in the cytosol. Some of them have an ER signal sequence that emerges at the N- terminus. This sequence that emerges is a hydrophobic AA sequence without any characteristic specific AA sequence, it’s mainly this hydrophobicity that gives it its characteristic ER signal sequence. For an ER protein, as it’s being translated from the ribosome, the ER signal sequence will emerge 1 at the N-terminus. - Once the ER signal sequence emerges from the ribosome, this whole complex is then directed to the ER membrane. As soon as the ER signal sequence emerges, it’s directed to the ER membrane, then the protein that is destined to go into the ER is directed through transmembrane transport in a co-translational process into the ER so the ribosome continues to translate the protein as it’s being funneled into the ER. - How this occurs is that there is actually this signal recognition particle cycle that occurs, that recognizes this ER signal sequence as it’s emerging from the ribosome so there’s a signal recognition particle that will bind to the ER signal sequence that will pause translation & then that whole complex with the signal recognition particle will be directed to a signal recognition particle receptor that’s on the ER membrane then translation will resume & the protein will be integrated into the ER in a co-translational process as the protein is being translated off the ribosome.  bind GTP  low affinity  high affinity - There are 2 major components to direct the emerging peptide with the signal sequence to the ER: the signal recognition particle also referred to SRP & the single recognition receptor SRP receptor. - Diagram: Here is the ribosome, the mRNA & the protein that’s destined for ER is being translated – the first thing to emerge is this N-terminal ER sndnal sequence – this will be bound by the SRP shown in the diagram & 2 this SRP will bind to the SRP receptor that is found in rough ER membranes – that will bring this entire complex to the ER. Both of these proteins have GTPase domains & they’re normally bound to GTP in this phase of the process where they’re bringing the ribosome & the newly synthesized protein to the ER. So they are both bound to the GTP so both the SRP & the SRP receptor have GTPase domains & are bound to GTP at this state. - Normally the SRP & the ribosome have a very low affinity for each other. When the ribosome is free without a protein coming out of it with the signal sequence, there is very low affinity b/w the 2 but when the SRP encounters a ribosome that has a protein with a ER signal sequence coming out of it then that becomes a very high affinity interaction & the SRP will bind to the signal sequence & then it can guide the protein & ribosome to the ER by binding to this SRP receptor.  Gated channel  Prevent diffusion of ions, small molecules  GTP hydrolysis Complex dissociates - In the ER, there is this protein translocator through which the newly synthesized protein will be funneled through. So you can think of it kind of like a tube of tooth-paste where the protein coming out would the toothpaste coming out of this ribosome which would kind of be the tube of toothpaste squeezing the protein out & injecting it into this protein translocator into the ER. - Diagram: The ribosome is brought to the protein translocator once it’s bound to the SRP receptor. Here is the ribosome with the newly synthesized peptide, the ER sequence here has bound to the SRP, SRP is bound to SRP receptor & now this entire complex is brought to this translocator. - The translocator channel is also called the translocon & it’s a gated channel meaning that it’s not always open – it’s kind of shown in the diagram as a plug but this will actually open once the ribosome & the ER signal peptide binds to the translocator. So what happens then is these 2, the SRP & the SRP receptor, bring the ribosome & the newly synthesized peptide to the translocator & the binding of the ribosome & the ER signal peptide to the translocon opens it up & allows the protein to be synthesized through the translocon. Now the ribosome forms a very tight seal with the translocator & this is important so it prevents the diffusion of ions & small molecules out of the ER. - Once this happens, how does this dissociation occur? What’s known is the SRP & the SRP receptor hydrolyze their GTP to GDP & inorganic phosphate & this somehow releases the complex so the SRP will be released into the cytoplasm & the SRP receptor will be released back into the ER membrane. It’s not known exactly what triggers this GTP hydrolysis at this time but what’s known is that GTP hydrolysis precedes the dissociation of the entire complex.  Start-transfer sequence  Cleaves ER signal sequence  Translocator gated in the 2 direction - Examples of how proteins are inserted into the ER. - Example of a soluble protein – this has no transmembrane domains & the 1 thing is that these proteins have an ER signal sequence at the N- terminus (again). All the translation is occurring in the cytosol & what is not shown here is the SRP & the SRP receptor but what you have to assume what has just happened is that the SRP & the SRP receptor have brought this newly synthesized protein to the translocon. - And in this case, the ER signal sequence acts as a start-transfer sequence. When the translocon binds to this N-terminal ER signal sequence, it actually signals this to start translocating that protein into the ER so this will bind directly so this signal sequence will bind to the translocon & that signals to the translocon to start transferring this protein through the translocon into the ER. So you can imagine up here (and it’s not shown either) is there is a huge ribosome that is actively translating this protein & as it’s translating, it’s being funneled into the ER. - So the start-transfer sequence or the ER signal sequence at the N- terminus binds to the translocator & then the protein is transferred into the ER. And then there is the signal peptidase & this is a protease that will cleave off the signal peptide off the protein so a mature soluble protein does not have its signal peptide associated with it b/c the signal peptidase will cleave the ER signal sequence once the protein gets inside the ER. What that does is it releases mature protein into the ER lumen & then the signal sequence that’s been cleaved off actually moves laterally nd into the lipid bilayer & that’s b/c this translocon is gated in a 2 direction so not only can it allow proteins to go through in this direction but it can also gate in the other direction also & allow proteins or this signal peptide to diffuse out into the membrane so it’s gated in 2 directions – one to allow the protein to enter the ER lumen & then the 2 direction to move things into the ER membrane. So the signal sequence will be cleaved off & then will be released into the ER membrane & the mature protein will be released into the ER lumen so this is for soluble proteins. - Answer: C - Answer: D - Animation shows how this transmembrane transport into the ER occurs. - This is for a soluble protein. Here is the ER, the signal sequence, signal peptidase cleaves it off then you have your protein soluble in the ER lumen.  Sorted out during translocation - Recall that there are different types: there are single-pass & multi-pass transmembrane proteins & this just means how many times the protein goes through the membrane – so a single pass will only go through the membrane once, while the multi-pass goes through the membrane multiple times. - Here are 3 examples of single-pass transmembrane proteins that are going through the ER membrane. Notice that in each of these 3 cases, there’s something different about the way they go into it – there are 3 different ways of inserting a single-stss trdnsmembrane protein into the ER membrane. In these 2 cases (1 & 3 in the diagram), the carboxy terminus of the protein ends up in the cytosol as compared to the 2 case where the carboxy terminus of the protein ends up in the ER lumen – this is due to the way in which the protein is inserted into the membrane.  Stop-transfer signal  COOH in cytosol - This example is very similar to soluble protein in that the protein has an ER signal sequence at its N-terminus. - Here is the ER signal sequence in the diagram & that will act as a start- transfer sequence – but in this case, unlike the soluble protein, there is another domain that acts as a stop transfer sequence – this is also a hydrophobic domain but since it comes after this start-transfer sequence as it’s being transferred through this hydrophobic domain will act as a stop-transfer & will actually compose the transmembrane domain. So this transmembrane domain is actually a stop-transfer sequence so what happens is the start-transfer sequence will start translocating the translocation of the protein, through the ER membrane, through the translocon & then this 2 hydrophobic domain is actually a stop transfer sequence – the transfer will stop there, the synthesis of the peptide will continue into the cytosol, the ribosome (not shown) continues to synthesize it on the cytosol side then the signal peptidase cleaves off the signal peptide & then the whole thing laterally diffuses into the ER membrane – so both the signal peptide & the mature transmembrane protein are laterally gated into the ER membrane – so both of these laterally diffuse into the lipid bilayer & what happens is in this case, the carboxy terminus ends up in the cytosol & the amino terminus ends up in the ER lumen. - This is the 1 way of getting a single-pass transmembrane protein into the ER membrane. - Animation: signal peptide there, there is the stop transfer stops there, continues into the cytosol, signal peptidase cleaves it & this time the lateral diffusion allows this to stay in the membrane. This is much more effective b/c you can see how the ribosome, the SRP, the SRP receptor are coming together to do this event. Note in this case, the carboxy terminus ends up in the cytosol & the amino terminus in the ER lumen.  Internal start-transfer sequence  Not cleaved  (+) amino acids on cytosolic face  COOH inndR lumen - This 2 example, in this case, you don’t have an N-terminus start- transfer sequence, it’s an internal start-transfer sequence. So the protein starts to be synthesized in the cytosol, the N-terminus comes out but it’s not right at the N-terminus that the start-transfer sequence occurs but rather internally. - Now one of the differences is this will not be cleaved off of the protein – an internal start-transfer sequence is not cleaved but this will also guide this protein to the ER membrane so the SRP will bind to this, bring it to the SRP receptor & then will start the transfer. - Once the transfer is complete, the translocon again gates in the lateral direction & allows this protein to laterally diffuse into the lipid bilayer. - In this case, the orientation is important here – what happens is the N- terminus is in the cytosol this time & the carboxyl terminus is in the ER lumen (reverse of what we just saw). What’s important is that there are positively charged AAs on one side of the start-transfer sequence & negatively charged AAs on the other side of this internal start-transfer sequence & the positively charged end of this internal start-transfer sequence always orients itself on the cytosolic face of the ER membrane so you can see here on the diagram that this end of the start-transfer sequence would be positively charged & would be facing the cytosol – that orientation means that the N-terminus will face the cytosol so what happens is as the protein is being synthesized, this internal start transfer sequence is exposed & will contortion itself in this way to make sure that these positively charged residues end up on the cytosolic face of the ER. Then what happens is the rest of protein is synthesized & pushed through the translocon until the carboxy terminus ends up in the ER membrane then the translocon will laterally gate to allow this transmembrane protein to move into the ER membrane. - So the orientation: the positively charged AAs of this internal start- transfer sequence are always on the cytosolic face & what this means in this case is that the carboxy terminus is in the ER lumen & the amino terminus is in the cytosol.  internal start-transfer sequence  not cleaved  N-terminus in lumen*  COOH on the inside* - Now we look at another example where we flip the charges on this internal start-transfer sequence – again there is an internal start-transfer sequence – this is the 3 example in terms of single-pass transmembrane proteins. There is an internal start-transfer sequence but this time the way it orients itself, note that the positive charges are towards the carboxy ter