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Lecture 19

Lecture 19

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

nd BIOL 300 October 22 2012 Lecture 19 Dr. Shock Remember, anything above 40 kDa in size is not able to freely diffuse across the NPC; anything below this size (like water and small molecules) can travel freely. mRNA and associated proteins are much larger than 40 kDa and therefore, there are specific mechanisms for its import and export. • Protein import is the simplest and one of the most common cases of transport; proteins are made in the cytoplasm, and proteins with functions in the nucleus must be imported. For a protein to be imported, it needs to have a nuclear localization signal, NLS, which is a short stretch of amino acids found almost anywhere in the protein. • In this example, pyruvate kinase, a protein which is normally found in the cytoplasm and not in the nucleus, can be seen through GFP tagging to be located only in the cytoplasm. • When they attached an artificial NLS to the pyruvate kinase and tagged it with GFP again, we can see that all of the protein is now in the nucleus. This shows that the NLS is both necessary and sufficient to get a protein across the NPC. We can see the process of protein import in this diagram; proteins which have an NLS are able to bind a transporter protein which contains FG repeats which are able to interact reversibly with the hydrophobic FG repeats nucleoporins. For example, a protein like pyruvate kinase with an attached NLS would interact with importin, an import protein. This complex is then able to DIFFUSE through the NPC into the nucleoplasm; this is not a form of active transport, it is driven by a concentration gradient. • One inside the nucleus, a Ran GTPase, when GTP bound, will bind to the importin- cargo complex causing dissociation of the cargo. • The importin-Ran-GTP is then able to be exported through the NPC, again by diffusion, into the cytoplasm, but the cargo is now stuck inside the nucleus. • The Ran-importing complex in the cytoplasm is then dissociated by a GAP, GTPase activating protein, through hydrolysis of GTP on Ran, causing a conformational change which causes importin to lose affinity for Ran. Also, in the absence of a GAP protein, the Ran-GTPase’s own intrinsic hydrolysis activity will be able to induce hydrolysis of GTP and release form importin. 1 nd BIOL 300 October 22 2012 Lecture 19 Dr. Shock • Then, Ran-GDP has to re-enter the nucleus where it can be re-attached to a GTP by a GEF (guanine nucleotide exchange factor) protein; GEF’s function is to kick the old GDP out and place a new GTP on the protein • The Ran is a small enough protein to be able to diffuse through the NPC by itself. • The whole cycle can then restart, and importin can bind to another protein. How do we achieve directionality of import; i.e. why does the diffusion of importin-cargo occur towards the inside of the nucleus and why does Ran-importin diffuse out of the nucleus? • This process works because of the localization of the GAP and GEF proteins; GEF is always in the nucleus, meaning active, or GTP-bound Ran will have much higher concentrations in the nucleus than in the cytoplasm, thus any cargo-importin entering the nucleus will be almost instantly dissociated. • Because of this quick dissociation, the concentration of the cargo-importin complex is higher in the cytoplasm than in the nucleus, causing net diffusion to be towards the nucleoplasm. • Similarly, GAP is always located in the cytoplasm, and therefore, cytosolic Ran-GDP concentration is always higher that its concentration in the nucleus. • This causes the Ran-GTP-importin complex concentration to be much higher in the nucleus than in the cytoplasm because of almost instant dissociation by GAP, driving the net diffusion of the Ran-GTP-importin complex out of the nucleus and into the cytoplasm. Some proteins also need to be exported from the nucleus to the cytoplasm, implying that they contain an NLS (they need to get to the nucleus in the first place), as well as another signal which allowed for export. These are known as shuttling proteins, which can move between compartments. This was discovered by fusing tissue culture cells from to different species (human and xenopus). To find potential exported proteins, they stained various nuclear proteins only in the human cells, and not the xenopus proteins. • In this experiment, they looked at two RNA binding proteins: hyRNPc and huRNPI; what they found was for huRNPc, only the human nuclei lit up, and there was no signal in the xenopus nucleus, as expected, because the human protein can’t get out of the human nucleus. • With the huRNPI, we see that the protein can be seen both in the human nuclei and the xenopus nuclei of the fused cells. This means; not only is the protein able to enter the xenopus nucleus, it must also be able to be exported from the human nucleus in order to reach the cytoplasm of the fused cell. One potential problem with the experiment above was that instead of old protein being exported form the nucleus, new huRNPI could have been translated in the cytoplasm and imported into the xenopus nucleus, avoiding the exporting mechanism altogether. • To prevent this, they introduced a translation inhibitor into the cells which 2 nd BIOL 300 October 22 2012 Lecture 19 Dr. Shock stopped the action of ribosomes,
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