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Lecture 7

BIO241 Lecture 7.doc

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Jennifer Harris

Tuesday, January 27, 2009 - Our continuation in the theme of movement within the cell – we’ve been looking at how proteins can move to different compartments in the cell & we looked at gated transport, transmembrane transport into the ER, for example & now we’re going to continue on with the 3 rd mechanism of transporting proteins around the cell called vesicular transport – compacting proteins into small vesicles & then moving them from one compartment in the cell to the another. - We started out looking at what the membranes in a cell looks like – the lipids & the proteins & we looked at how these membranes are selectively permeable to certain molecules. Proteins, for example, can’t freely diffuse across membranes b/c they’re impermeable to a large hydrophilic or ions, anything with a charge & the further you go away from hydrophobicity, the less permeable the membrane is those molecules so you need active mechanisms to transport these ions & proteins across membranes. - We looked at how small molecules can be transported by these transporter proteins or these channels in order to move from one compartment to another or into and out of the cell. - Then we also looked at how proteins are sorted to the nucleus by gated transport, to the ER by transmembrane transport, to the mitochondria. - Today, we’re going to look at how proteins are transported by vesicle trafficking. - This is a continuation of what goes on in the ER.  Found in non-cytosolic leaflet **Don’t need to know the structures of these lipids** - The majority of lipids are synthesized in the ER of a cell – these include phospholipids, cholesterol & another lipid called ceramide. - A certain subset of these will be modified, in particular ceramide will be modified in the Golgi Apparatus. Ceramide, which is synthesized in the ER will be used as a precursor for 2 particular lipids in the Golgi: sphinogomyelin (SM) where the polar head group from phosphocholine (PC) is transferred onto ceramide & then glycolipids are also synthesized in the Golgi & this is when sugars or oligosaccharide chains will be added onto lipids & this is the synthesis of glycolipids. So both of these are synthesized in the Golgi from a ceramide lipid precursor & the ceramide is synthesized in the ER along with these other abundant lipids in animal cells. - These ones that are synthesized in the Golgi will be found in the non- cytosolic leaflet of the plasma membrane since they are synthesized in the lumen of the Golgi – so the non-cytosolic leaflet is the one exposed to the extracellular space. These ones in the Golgi are synthesized in the lumen of the Golgi. So this is where the majority of membrane lipids are synthesized.  by ER expansion  new lipids in cytosolic leaflet of ER. - New membranes are synthesized by ER membrane expansion which means that lipids are synthesized & added into the membrane of the ER. Specifically, the synthesis occurs on the cytosolic leaflet of the ER. Diagram: all the enzymes that catalyze the synthesis of these lipids have their active site facing the cytosol – so that means all the lipids are being synthesized on the cytosolic leaflet of the ER (not going to go into details of all the enzymes or reactions that occur to synthesize lipids). All these enzymes have their active site on the cytosolic side of the ER membrane & the lipids are being synthesized in this leaflet. - This presents a conundrum to the cell – if the synthesis were just to occur continuously on this leaflet of the ER membrane, you would just get expansion of this membrane whereas the other membrane wouldn’t expand. Recall that lipids don’t tend to flip flop from one leaflet to another on their own unless there are enzymes there to catalyze that flip- flop from one leaflet to another. They would just continuously accumulate in the cytosolic leaflet & then you would get expansion of one leaflet & the other one would not expand at the same rate. So to overcome, the cell has specific enzymes that will flip lipids from the cytosolic leaflet into the luminal or non-cytosolic leaflet of the ER membrane.  rapidly flipped to other leaflet  scramblase  by vesicular transport - Diagram: This would be the ER membrane, it’s a lipid bilayer & then you’re getting new lipids synthesized on the cytosolic leaflet & you can see that this leaflet would expand & if there was no enzyme to flip it, this leaflet would expand but this other one would not so there is a specific enzyme called the scamblase that catalyzes the flipping of phospholipid molecules. And it will flip specifically lipids to equilibrate them b/w the 2 leaflets of the membrane so it doesn’t preferentially flip from one leaflet to another, it just basically equilibrates lipids b/w these 2 membranes & this is not specific so it will do this for almost all phospholipids that are synthesized in the ER membrane & equilibrate them b/w the 2 leaflets of the membrane. Scramblase will make sure that there is a symmetric distribution of lipids in the ER membrane so only one leaflet doesn’t grow preferentially over the other. This leads to the symmetric growth of both halves of the bilayer. - Then from there, the newly synthesized membranes will be distributed to other membranes in the cell, such as the Golgi, the plasma membrane & the endosomes – they will be distributed to these ones from vesicular transport. These vesicles are made up of membranes themselves & they originate originally from the ER so they’ll go from the ER to the Golgi to different compartments in the cell & with them they carry along lipids & distribute them to these other membranes.  Flippase (ABC transporter)  energy from ATP hydrolysis - Recall there are also specific lipids that are found in certain leaflets of membranes & in particular, the plasma membrane has specific lipids on its cytosolic leaflet, & these include phosphatidylserine (PS) & phosphatidylethanolamine (PE) – these are preferentially found on the cytosolic leaflet of the plasma membrane. - So if the scramblase is scrambling these lipids from one leaflet to another, how does the cell preferentially enrich a certain lipid into the cytosolic leaflet of the plasma membrane? This is done through another phospholipid translocator & this one flips specific phospholipids to the cytosolic leaflet of the plasma membrane – called a flippase – it is an ABC transporter which transports lipids from one leaflet of the membrane to another. - Diagram: Here would be a newly delivered membrane going to the plasma membrane – this would come from the ER – you can see that the lipid is randomly distributed b/w the 2 leaflets, the yellow & the red colours but now let’s assume that the red one would be PS, for example – once it associates with plasma membrane, it needs to be preferentially enriched on the cytosolic leaflet of the plasma membrane so this is carried out by this flippase that will flip preferentially these PS to the cytoplasmic leaflet of this lipid bilayer. This is very important b/c the distribution of PS to the non-cytoplasmic leaflet of the plasma membrane can be an indication that a cell is dead so when cells die these flippases turn off, PS become enriched in non-cytosolic leaflet which is not their normal situation & this enrichment of PS now in the non- cytosolic leaflet can be a signal for macrophages to come & digest & engulf that cell. So it’s very important that a cell that is healthy keeps the PS on the non-cytosolic leaflet to prevent macrophages from engulfing & digesting that cell. - So that’s the way that lipids are distributed in the membranes: 1 st they’re synthesized in the ER, scrambled b/w the 2 leaflets & then delivered to the different compartments through vesicular transport & then if some lipids need to be enriched in one leaflet over another, flippases will specifically flip those lipids to the appropriate leaflet of the plasma membrane.  budding with cargo  fusion to target  release cargo - Vesicular transport occurs from the ER to the Golgi, from the Golgi to the cell exterior through a process called exocytosis, from the Golgi to endosomes (intermediate compartments b/w the Golgi & the lysosome or the Golgi & the cell exterior, for example), & also there’s a process called endocytosis where vesicles can come from the plasma membrane & come into the cell to these endosomes & uptake nutrients or other molecules from the outside of the cell. This movement of proteins occurs through vesicular transport. - So whatever is inside a vesicle is referred to as cargo. - Diagram: There are 3 major components to this process. One of them is the budding of a vesicle with cargo from a donor compartment so let’s assume this is where the vesicle is originating from, the vesicle has to bud off this donor compartment, it could be the Golgi for example, or the ER, & when it buds off, it needs to contain cargo. This cargo is usually made up of some proteins & these proteins can either be soluble proteins or transmembrane proteins. So the vesicle needs to bud off the donor compartment completely & then it needs to fuse with a target compartment so the donor compartment could be ER & the target compartment could be the Golgi, for example. So vesicle buds off with its cargo & fuses with the Golgi & then it needs to release its cargo into the target compartment. So 3 major steps to delivering a protein to a new compartment through vesicular transport. - This is just to show you the different processes where vesicular transport is important for transporting macromolecules inside the cell. This is called intracellular vesicular transport & the different arrows refer to different types of transport that is occurring. - The red arrows include the secretory pathway & this is usually movement that originates from the ER early, through the Golgi, through to the early endosomes or lysosomes & it also includes movement to outside of the cell through exocytosis. So its movement is from the ER, through to the outside of the cell through intermediate compartments such as the Golgi & lysosome & endosomes – that would be the secretory pathway. - The green lines indicate the endocytic pathway which is the formation of vesicles on the plasma membrane & uptaking macromolecules from outside of the cell & the endocytic pathway goes from the plasma membrane to the early endosome & usually ends up in the lysosome. - Now in blue you have retrieval pathways to ensure that lipids don’t over-accumulate in one area or another so if movement was always going in this direction, lipids would be lost to the ER & accumulate in plasma membrane & the plasma membrane would continue to grow until the cell would be too large for itself. So there needs to be retrieval pathways to recover membranes that are being moved in a certain direction so the blue arrows indicate what these retrieval pathways are so as vesicles are moving through the secretary pathway from the ER to the Golgi, there are also retrieval pathways to recover some of these lipids through vesicles also back to previous compartments in the Golgi & also back to the ER. So retrieval pathways can also come from the early endosomes, late endosomes & so on & you can see that there are also retrieval pathways to recover lipids to the plasma membrane – there is endocytosis occurring so that the lipids are not all lost from the plasma membrane – lipids are being recovered through a retrieval pathway from the early endosomes. - So these are 3 major types of intracellular vesicular transport that are occurring – all of these use vesicles for transport.  Transmembrane proteins  Soluble proteins - This is just an outline again to show you how this vesicular transport occurs. This figure is nice b/c it shows you the lipid bilayer in 2 different colours & you can see how this lipid bilayer is forming into a vesicle & then note how the luminal side of the donor compartment forms the inside of the vesicle & then when you fuse to the target compartment, the luminal side/leaflet of the bilayer also becomes the luminal leaflet of the new compartment so the orientation of these leaflets is maintained within this transport. - If we were to envision this as the plasma membrane so the target compartment wouldn’t be closed off, this would be the plasma membrane you can see that the luminal leaflet (the blue one) would actually become the extracellular leaflet of the plasma membrane so let’s assume this would be the cytosol & this would be the extracellular space, this blue leaflet now becomes exposed to the extracellular space & this explains why things that are occurring on the luminal side of the leaflet end up on the outside of the cell. - When a vesicle buds, cargo proteins need to be collected into that vesicle & these can be transmembrane proteins (the green ones in the diagram) but they can also be soluble proteins & many of these soluble proteins are actually bound by transmembrane cargo receptors. So these are transmembrane proteins that will bind to the soluble proteins & recruit them to the vesicle that is forming. - Then once the vesicle is loaded with the cargo, it will bud off & then fuse with the target compartment.  select cargo for vesicle  give curvature to vesicle - Newly emerging vesicles off a compartment have protein coats over them. There are 3 major types of protein coats: clathrin coats, COPI coats, & COPII coats. - Now clathrin coats are mainly coats that are on vesicles forming from the plasma membrane or from the Golgi & going to endosomes. - COPI coats are mainly going within the Golgi so vesicles going from one compartment of the Golgi to another or vesicles coming from the Golgi to the ER. - COPII are on vesicles that are forming on the ER & going to the Golgi. - The function of these protein coats is to select the cargo for these vesicles, to give curvature to the vesicle which aids in the formation of the vesicle as it’s forming off the compartment & to promote the vesicle budding. These coat proteins play an important role in selecting what’s going to go into a vesicle & then actually inducing the formation of the vesicle.  from Golgi to ER (indicated in blue here)  b/w different Golgi cisternae (so within the Golgi from one compartment of the Golgi to the other)  from ER to Golgi (indicated in red)  from Golgi apparatus (to the endosome) & plasma membrane to endosome (indicated in green) - So the different types of protein coats are indicative of where these vesicles came from & where these vesicles are destined to go. - So how do these protein coats form on the newly emerging vesicles? One important player in this is these monomeric GTPases so we saw these GTPases when we looked at gated transport into the nucleus. GTPases also play an important role in recruiting protein coats to newly forming vesicles. - The activity of these GTPases is regulated by 2 classes of protein: GEF – exchange the GDP bound GTPase ndr GTP so you can think of it as activating that GTPase & the 2 class of regulators is GAP – activates the GTPase activity of this enzyme & the enzyme will hydrolyze the GTP to GDP & then what happens is you can consider that to be turning off the GTPase. So GEFs activate & the GTPase is bound to GTP & GAPs inactivate by converting the GTP, inducing the conversion of GTP-bound form to GDP-bound form.  Vesicle ready for transport to target compartment - This is a generic overall of how these GTPases actually promote coat assembly. - Diagram: There is a GEF (the one that will activate GTPase) that’s found in the membrane where the vesicle is going to bud & that will recruit the GTPase to the membrane, exchange the GDP to GTP & this will activate this GTPase. In this case, when the GDP is exchanged for GTP, this GTPase will associate with the membrane where the vesicle is going to form. So basically you have a GTPase that’s recruited to the membrane where the vesicle is going to form by a GEF that’s found in that membrane & that will activate the GTPase & the activated GTPase will now associate with the membrane where the vesicle is forming. - Once the GTPase is activated, it’s bound to the GTP-bound form & it will recruit the coat proteins. In this case, this little yellow & brown structure would be the GTPase & it will recruit the different coat proteins shown here in red & this would be the outer coat proteins so basically it’s recruiting this coat to the area where the vesicle is going to form. - Once the coat forms, this will induce vesicle formation so the membrane will start to curve & the vesicle will form & eventually bud off the membrane. Once the coat is recruited the coat will 1 of all recruit the cargo to that vesicle, the vesicle will bud off & the final thing that’s important is that once the vesicle buds off the donor membrane, the coat will be shed before the vesicle will fuse to its target membrane so the vesicle buds off, the vesicle is un-coated so the protein coat falls off & for COPI & COPII coated vesicles, this involves GAPs & for the clathrin coat, there is a different mechanism that we’ll look at. - Once these vesicles have shed off their coat, then they’re ready for transport to their target compartment but remember that the coat has to be shed off the vesicle before it can fuse to its target compartment.  ARF GTPase  Sar1 GTPase  recruits Sar1  recruit coat protein subunits - So let’s look at a specific example here. - For COPI & for clathrin coated vesicles, the GTPases are called ARF GTPases & these are the ones responsible for recruiting these 2 types of coats, the COPI & the clathrin coats to these vesicles. - For COPII coats, it’s the Sar1 GTPase that recruits these COPII coats & this is shown on the right diagram how this occurs. So here is the Sar1 GTPase in the cytosol, it’s not associated with the membrane & it’s bound in its GDP-bound form so this would be considered the inactive form. It actually has an amphipathic helix that allows it to associate with the membrane where the vesicle is going to form but it’s hidden inside the protein & not exposed in the GDP-bound form. - There is a Sar1 GEF that’s found in the membrane where the vesicle is going to form that recruits the Sar1 GTPase to the appropriate membrane where the vesicle is going to form. The GEF will exchange the GDP for GTP on the Sar1 GTPase & the binding of GTP to the Sar1 GTPase exposes this amphipathic alpha-helix & that allows it to associate with the membrane through this amphipathic helix so the GTP-bound form will bind to the target membrane. - So just to over in more detail, the Sar1 GEF is in the ER membrane. Recall that COPII vesicles are moving from the ER membrane to the Golgi, so in the ER membrane, this GEF has to recruit this Sar1 GTPase to initiate the vesicle formation. So the Sar1 GEF recruits Sar1 (as shown in the slide) – it will activate Sar1 by exchanging the GDP for GTP & once this happens, an amphipathic helix is exposed & allows it to interact with the membrane. And once Sar1 GTP is in the membrane, it can recruit coat proteins to that membrane & then the coat proteins are the ones that will recruit the cargo & induce the vesicle formation.  cargo (these will be transmembrane proteins)  transmembrane cargo receptors (the ones that will bind to soluble cargo proteins)  SNAREs - Diagram: Inner layer is shown here, can see the Sar1 GTPase but it also recruits these coat proteins that are in the inner layer – this inner layer is mainly responsible for binding to the membrane of the donor compartment & also, it will select the cargo that’s going to be in the vesicle. -The outer layer of the vesicle coat associ
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