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BIO241 Lecture 5

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Jennifer Harris

- We’re going to look at sorting of proteins into different intracellular compartments. - We’ve been looking at the movement of small molecules across the different membranes in a cell but the proteins also need to move to the different compartments in order for the compartments to have their specific functions. For example, the ER, the Golgi, the peroxisome and the nucleus all need specific proteins in order to maintain their proper functions. For example you can imagine that a transcription factor needs to get to the nucleus in order to transcribe DNA into RNA so that transcription factor needs to be sorted to the nucleus in order to function properly. - Again the 1 lecture we mainly went over membrane components & looked at the transport of small molecules across these membranes & now we’re going to look at the sorting of proteins across these membranes into the different compartments within a cell. For example, the mitochondria & we’re going to look at cytosolic proteins, the peroxisomes, the ER, & of course the nucleus. We’re also going to look at the nucleus & how proteins also get into & out of cells. - For today, we’re going to look at protein sorting into the nucleus, mitochondria and into the peroxisomes.  Within cell from different compartments  Out of cell  Into cell - Now we can look at the movement of proteins. Don’t forget that the membrane surrounds the cell itself, the PM and also the organelles within the cell. The peroxisomes are surrounded by a membrane, the nucleus is actually surrounded by two lipid bilayers, mitochondria also have a lipid bilayer surrounding them, the Golgi, the endoplasmic reticulum, all of these are compartments which contains proteins and need proteins sorted to them. - We can look at movement of proteins within the cell from different compartments, we’re also going to look at the movement of proteins out of the cell across the plasma membrane and then how do cells uptake proteins from the extracellular environment in a process termed endocytosis. - So how do proteins get from outside the PM to the inside of the cell? There are thousands of proteins synthesized in a cell and the protein synthesis is all initiated on ribosomes in the cytosol. Initiation if you’ll recall from BIO240 in the fall, you will recall that DNA is transcribed into RNA, the RNA is shuttled into the cytoplasm where it is translated by ribosomes into proteins. All proteins, the initiation of translation are all initiated in the cytosol. - From the cytosol, proteins need to be sorted to their proper locations. An ER protein needs to be translocated from the cytosol to the ER and to do this, the proteins have certain signaling sequences on them that guide them to the proper locations. The amino acid sequence contains a peptide or a signature sequence that will guide the proteins to their appropriate compartments. - So just appreciate the thought that protein synthesis of almost all proteins is initiated in the cytosol.  Proteins fully synthesized in cytosol before sorting  Associated with ER during protein synthesis - The different types of protein sorting can occur post-translationally, so in a post-translational process, the proteins are fully synthesized in the cytosol before being sorted. The entire protein is made and then it is sorted. This occurs for sorting to the mitochondria, the plasmids or chloroplasts, nucleus and to the peroxisomes. - There are two different types. In one type, the protein remains unfolded. There are usually specific proteins called chaperones that maintain these proteins in the unfolded state before they’re sorted to the mitochondria or to the chloroplast. In the second type, the proteins are completely folded after their translation and then the folded protein is translocated into the nucleus or to the peroxisomes. In both cases, these occur post-translationally. - In contrast, there is also co-translational sorting that occurs and this occurs into the endoplasmic reticulum. So proteins with an ER signal sequence will associate with the endoplasmic reticulum during protein synthesis. - We’re going to go into details about this in the next lecture but basically, this just shows us what this kind of co-translational process looks like, here would be the ribosome, the mRNA is being translated into protein and as the protein is being translated, the protein is being funneled into the ER across the ER membrane. This is sorting in a co-translational manner. As the protein is being translated, it is being sorted to the ER, you can see that the ribosome is closely associated with the ER and that is what gives the appearance of the rough ER, is the setting of ribosomes all around the ER. - There are three main types of transport that we’re going to cover. - Gated transport which refers to the movement of proteins from the cytosol to the nucleus. - Transmembrane transport which occurs to the endoplasmic reticulum as we just saw in a co-translational process, but it also occurs to the mitochondria, to the plastids and to the peroxisome. Usually, translocator protein complexes are involved in the process of transmembrane transport. - And then the third kind is vesicular transport, where vesicles move proteins between the compartments. We’ll see examples of this when proteins move from the ER to the Golgi and when the proteins move from the Golgi to the endosomes or the lysosomes and also for proteins that are bound for the outside of the cell in secretory vesicles.  Selective transport of macromolecules  Free diffusion of small molecules (<5000 Daltons) - The first type is called gated transport and this refers to the movement of proteins between the cytosol and the nucleus. This occurs through what is called the nuclear pore complex. The nuclear pore complex traverses the two membranes of the nucleus, so if you’ll recall the nucleus has two lipid bilayers surrounding it and the nuclear pore complex goes through both of those membranes and forms a pore. - This pore is gated meaning that it is selective for the transport of macromolecules, it is called a pore but it is selective for certain macromolecules. However, if molecules are small enough, less than 5000 Daltons, they can freely move between the nucleus and the cytosol and the nuclear pore complex is no longer selective against these molecules. Anything above 5000 there is selective transport by the nuclear pore complex and anything below 5000 can freely diffuse through the nuclear pore complex into the nucleus or out of the nucleus.  From cytosol to nucleus  From nucleus to cytosol - The nuclear pore complex can accommodate transport in both directions, that means into the nucleus or out of the nucleus. - Here is a diagram of a nuclear pore complex. There are approximately 30 proteins called nucleoporin that make up the nuclear pore complex, and this, the whole complex you can see that it goes through both the outer nuclear membrane and the inner nuclear membrane. Remember both of these would be lipid bilayers, so there are two lipid bilayers that make up the nuclear envelope that surrounds the nucleus. - The nuclear pore complex can accommodate transport in both directions. Nuclear import is the movement from the cytosol to the nucleus and nuclear export is the movement from the nucleus to the cytosol. - Some proteins need to move into the nucleus such as transcription factors following their synthesis or their translation into the cytoplasm and then some proteins need to actively be exported out of the nucleus so this can also be some proteins that need to go into the nucleus sometimes but need to be exported at other times during a certain event. Or you can also have ribosomal proteins that are assembled in the nucleus and need to be translocated into the cytosol in order to participate in translation. There are proteins that need to be actively exported outside of the nucleus.  Binds to NLS (rich in Lys and Arg – these are positively charged AAs)  Binds to nucleoporins in NPC - The import of proteins into the nucleus involves cargo proteins. The cargo proteins are what are being delivered into the nucleus. It possesses a nuclear localization signal or what is abbreviated as NLS. Nuclear localization signal is a specific AA sequence on a cargo protein that will guide it or allow it to be imported into the nucleus. Specifically, a nuclear import receptor will bind to the nuclear localization sequence. The nuclear localization sequence is a sequence that is rich in lysine and arginine amino acids. These are positively charged amino acids. - Once the NLS has bound to the nuclear import receptor, it binds to the nucleoporins in the nuclear pore complex and then the cargo protein will be transported into the nucleus. - Here we have the cargo protein that is destined for the nucleus, it has the nuclear localization signal rich in lysines and arginines, it will bind to a nuclear import receptor and this nuclear import receptor carrying the cargo protein will move into the nucleus. - You can fuse different nuclear import signals to different proteins and they’ll also go into different nuclear import receptors so you can swap. If you add this protein or this NLS onto another protein that doesn’t normally have a nuclear localization signal, you can guide it to go into the nucleus. This NLS is sufficient to guide a protein into the nucleus.  Binds to NES  Binds to nucleoporins in NPC - Similarly, export of proteins out of the nucleus, the proteins that are going to be shipped out of the nucleus are called cargo proteins. But in this case, you have a nuclear export signal unlike the nuclear localization signal, it is the NES nuclear export signal and he gave the examples of certain ribosomal subunits need to be exported, RNA that is transcribed in the nucleus need to be exported out, proteins with regulated nuclear import and export will also have nuclear export signals on them. - Similarly, the NES will bind to the nuclear export receptor. This is structurally related to a nuclear import receptor but instead it binds to an NES and then it binds to the nucleoporins in the nuclear pore complex and transports the protein into the cytosol. Again you have a protein with an NES, this would be the NES there, it can bind, this would be referred to as the cargo protein. The cargo protein would bind to the nuclear export receptor through its NES and this would be exported into the cytosol. Both the cargo protein and the nuclear export receptor would be transported from the nucleus to the cytosol. - Here is an overview of this process. On the left there you have nuclear import, on the right, nuclear export. - So cargo is being delivered in this case from the cytosol there to the nucleus and you have the cargo there with the NLS, it binds to a nuclear import receptor, both of them the cargo and the import receptor move into the nucleus through the nuclear pore complex. - Now what happens here is Ran-GTP is a GTPase that is required for this nuclear import process. What will happen is Ran GTP will bind to the receptor, that will release the cargo and the Ran GTP bound to the receptor will move back to the cytosol that way the nuclear import receptor can be recycled back to the cytosol. Once in the cytosol, the GTP that is bound to Ran will be hydrolyzed to GDP so it becomes Ran GDP. - We are going to go into more detail about how this actually happens but basically just appreciate that Ran GTP plays an important role in the nuclear import process. - Similarly in the nuclear export process, here is the cargo protein with its nuclear export sequence and in this case, the Ran GTPase bound to GTP will bind together with the cargo protein to the nuclear export receptor. And then this complex of Ran GTPase with the cargo and the nuclear export sequence, nuclear export protein will all move to the cytosol through the nuclear pore complex. What happens now is that Ran GTP, the GTP is hydrolyzed to Ran GDP, it is released and so is the cargo and in this case, the nuclear export receptor can just cycle back to the nucleus. - From this slide it is important to recognize that in both export and import, the Ran GTPase is required.  GDP bound form  GTP bound form  Stimulates GTP hydrolysis by Ran  Promotes exchange of GDP for GTP by Ran  In cytosol  In nucleus - The Ran GTPase cycles between two forms, the GDP bound form and the GTP bound form. You can see the GDP bound form in the slide and the GTP form as well in the nucleus. - The Ran GTPase is regulated by two proteins. One is called Ran GAP – GAP stands for GTPase Activating Protein. What this does is it stimulates GTP hydrolysis by Ran. This results in Ran being bound to GDP. The Ran GTP is in the slide, it moves out into the cytosol, Ran GAP activates the GTPase function of Ran GTPase so Ran GTPase hydrolyzes GTP and you get Ran GDP. So the Ran GAP is localized here in the cytosol and it activates or stimulates GTP hydrolysis by Ran. - On the other hand, in the nucleus you have Ran GEF which is a Guanine nucleotide Exchange Factor and basically what this does is it promotes the exchange of GDP for GTP by Ran. You’ll have Ran GDP coming into the nucleus, it is acted upon by Ran GEF and then the GDP is exchanged for GTP and you end up with Ran GTP bound in the nucleus. - Now the differential distribution of these two regulatory proteins is critical for maintaining the levels of Ran GTP and Ran GDP. Now Ran GAP is found in the cytosol and Ran GEF is found in the nucleus. What happens is you get a high Ran GTP concentration in the nucleus b/c Ran GEF is constantly exchanging the GDP for GTP so the Ran GTPase is bound to a GTP in the nucleus. However in the cytosol, Ran GAP is continuously stimulating the GTPase activity of Ran GTPase, this results in GTP hydrolysis by Ran and Ran GTPase becomes bound to GDP. So Ran GAP is converting Ran GTP to Ran GDP and this is occurring in the cytosol. So that results in a high Ran GTP concentration in the nucleus and a low Ran GTP concentration in the cytosol. This is critical for the direction of transport as we’ll see. - So Ran GTP we just mentioned that it is important for the direction of transport and that is because Ran GTP moves to the cytosol with the nuclear import and export receptors so this is shown in the slide. - Let’s say this is a nuclear import receptor, once Ran GTP binds, there would be an empty import receptor, when it binds to Ran GTP it is exported back to the cytosol. There would be an export receptor in the slide there and when Ran GTP binds with the cargo, it also exports out into the cytosol so in both cases Ran GTP binding to the import or the export receptors result in the movement of these receptors from the nucleus to the cytosol. - Ran GTP once it gets into the cytosol, it gets acted on by Ran GAP which leads to hydrolysis from GTP to GDP and you’ll end up with Ran GDP. Now what happens is Ran GDP is cycled back to the nucleus and this involves another protein called nuclear transport factor 2 or NTF2 shown in the slide which is responsible for recruiting or transporting Ran GDP back to the nucleus from the cytosol. Ran GDP moves back to the nucleus with the help of NTF2 and then in the nucleus, Ran GEF will exchange the GDP for GTP and then you get Ran GTP again and then this cycle can continue where Ran GTP will bind to the nuclear import and the nuclear export receptors. - Ran GDP and GTP are continuously moving back & forth b/w the nucleus & the cytosol.  Binds cargo in cytosol  Causes cargo release  GTP hydrolysis  Release of import receptor - Now let’s look at nuclear import of cargo in greater detail. - The first event occurs in the cytosol, there is an empty nuclear import receptor, there is also a cargo protein with a nuclear localization sequence. First event, the nuclear import receptor binds to the cargo, the receptor and the cargo move to the nucleus through the nuclear pore complex. - Now once in the nucleus this is where Ran GTP comes in, Ran GTP binds to this complex and this causes release of the cargo shown in the slide. Ran GTP takes the place of the cargo causing the release of the cargo and binds to the nuclear import receptor. - Now once this is done, the cargo is now in the nucleus, the receptor has to be recycled back to the cytoplasm so the empty import receptor with the Ran GTP now moves to the cytosol. - So then what happens in the cytosol is that Ran binding protein, this we didn’t introduce but there is another protein involved in this association complex here which is called Ran binding protein and Ran GAP which will lead to the hydrolysis of GTP to GDP, both promote GTP hydrolysis of the Ran GTP and release of the nuclear import receptor. Once this complex arise in the cytosol, it is acted upon by two proteins, Ran binding protein and the Ran GAP and both of these promote GTP hydrolysis so you end up with Ran GDP and that releases the import receptor. Then what you get is an empty nuclear import receptor that is free to bind to another cargo molecule and continue this process. You can see this cycling. - That was the nuclear import process so this animation will be available on the Bio241 website you’ll want to take a look at it.  Binds Ran-GTP + cargo in nucleus  GTP hydrolysis  Release of cargo  Release of export receptor - That was nuclear import so now we cover nuclear export which follows very similar principles, in this case though you use a nuclear export receptor rather than a nuclear import receptor. - We
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