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NUCLEAR IMPORT AND EXPORT

Some Notes to go with the video (recommended to read after the video) Protein sorting between the nucleus and the cytosol is termed gated transport, with the gate being the nuclear pore complex, or NPC. The nucleus has a double membrane surrounding it called the nuclear envelope. The nucleus allows passive entry and exit of small molecules (smaller than 5000 daltons) but controls the passage of macromolecules like proteins, many of which are in the range of ~25,000 daltons, as well as DNA and RNA polymerases, many of which are around 100 – 200 THOUSAND daltons. Ribosomes, which are huge, actually get assembled in the nucleus and must then be exported – being 30 nm across, they can’t get back in from the cytosol. The nuclear pore complex contains channel nucleoporins with large unstructured regions creating a messy tangle. This is why macromolecules can’t freely come and go but small molecules can. In order to enter the nucleus, a macromolecule needs a nuclear localization signal, or NLS, and in order to exit, it needs a nuclear export signal, or NES. With active transport, molecules up to 39 nm across can be carried through the nuclear pore complex. Protein cargo destined to be transported into the nucleus has a Nuclear Localization Signal (NLS) rich in positively-charged Lysine and Arginine amino acids, which allows it to bind to a Nuclear Import Receptor, also called an Importin. Different cargo proteins can have different nuclear localization signals and can therefore bind different importins. An adaptor is sometimes necessary to help the importin bind the cargo. The Importin binds to nucleoporins, at binding sites with many phenylalanine glycine repeats termed FG-repeats, in the Nuclear Pore Complex and is transported into the nucleus. The Nuclear Export Signal on macromolecules destined to leave the nucleus has a different amino acid sequence than the nuclear import sequence. The nuclear export receptor also binds to nucleoporins in the nuclear pore complex and is similar in structure to the nuclear import receptor. In fact, they are both encoded by a gene family called the karyopherins, or nuclear transport receptors. Now, let’s take a look at how nuclear import works A macromolecule has a nuclear localization signal and is hence destined to be transported into the nucleus. It binds to an importin and the bound importin binds to nucleoporins of the nuclear pore complex. The cargo-bearing importin is shuttled across. At the other end of the nuclear pore complex, there is a Ran GTP. Ran GTP binds the importin, which has a sort of stacked alpha helix motif forming a spring-like structure. The spring-like structure has a conformational change and the cargo protein is released into the nuclear lumen, which is its destination. After this exchange, the importin bound to Ran-GTP is transported back into the cytoplasm. Ran-GTP is hydrolyzed to Ran-GDP and inorganic phosphate and dissociates from the importin. The importin can now be reused to transfer the next cargo. For simplicity’s sake, I omitted a few details in the previous explanation. There are a few more players involved in nuclear import (and also export). Firstly, Ran is inefficient at GTP hydrolysis. And so Ran Binding Protein (RBP) – not shown here – and Ran GAP must work together to hydrolyze GTP and release the nuclear import receptor. An easy way to remember Ran-GAP is to think of it as… LITERALLY… creating a GAP where there used to be a phosphate group. Secondly, we have Nuclear Transport Factor 2, or NTF2 for short, which transports Ran-GDP into the nucleus. Inside the nucleus, we have Ran-GEF (Guanine Exchange Factor). Ran-GEF is bound to chromatin, and catalyzes Ran to exchange GDP for GTP. Upon completion, NTF2 returns to the cytoplasm. Here’s an brief overview of import. Feel free to pause the video and read. Nuclear Export is very similar to Import so I’m going to cover it quickly. A nuclear export receptor binds to nucleoporins and is transported into the nuclear lumen. There it binds with Ran-GTP, which then promotes binding with its nuclear export signal-bearing cargo. The receptor again binds to nucleoporins and the whole thing is transported to the cytosol. Ran-GAP helps with hydrolysis of Ran-GTP to Ran GDP and Ran Binding Protein helps detach Ran from the receptor. The cargo dissociates and the nuclear export receptor is free. Now, the receptor returns to the nucleus without any enzymes helping it.