School Papers

Surrounding and cytosol. However, certain nuclear activities require

Surrounding the genome is the nuclear envelope, which composed of two membrane layers – the inner and outer. The amphipathic layer prevents substances from travelling freely between the nucleus and cytosol. However, certain nuclear activities require materials obtained from the cytoplasm to occur. Same goes to cytoplasmic activities such as transcription which require synthesized RNA molecules from nucleus to proceed. These molecules must travel through a channel called nuclear pore complex (NPC) to get to the destined location.NPCs are made of a protein family of 30 called nucleoporins. NPC contains 500-1000 nucleoporins. They are imbedded into the nuclear envelope penetrating through both membranes allowing substances to surpass the hydrophobic regions. The centre of the NPC consists of hydrophilic passageways allowing small water-soluble proteins (<5000 Daltons) to pass through freely. These passageways were created by the disordered region of channel nucleoporins baring numerous hydrophobic phenylaniline-glycine (FG) repeats, acting as a sieve, stopping larger proteins (>60000 Daltons) from entering. Proteins such as DNA polymerase require a Nuclear Localising sequence (NLS) located anywhere on the protein surface.NLS is composed of a string of positively charged, basic amino acids lysine and arginine which sequence varies among proteins. The more NLS, the faster the rate of transport. This sequence is complementary to the binding site on the ?-Karyopherin. Karyopherin, also known as importins or exportins are soluble transport factors that allows movement of proteins across the nuclear membrane. The NLS on protein (cargo) interacts with central NSL binding domain on ?-Karyopherin bound to ?-Karyopherin. The binding between ? and ? karyopherin forms a heterodimer, causing a higher affinity for substrates compared to ?-Karyopherin alone. This blocks the N-terminus of ?-Karyopherin stopping it from autoinhibiting the central NSL binding domain (Chook & Blobel, 2001).  ?-karyopherin contains FG repeats, hence interacting weakly with cytosolic fibrils. However, the real work here is by binding and dissociation between ?-Karyopherin and the disordered region, moving it from chain to chain.heterodimer to disband. The binding sites of RAN-GTP and ?-Karyopherin are overlapping as shown in figure 1. RAN-GTP would occupy that region by displacing ?-Karyopherin as it has a higher affinity, blocking the binding site for ?-Karyopherin (Moroianu, Blobel & Radu, 1996). The cargo on ?-Karyopherin is displaced by cellular apoptosis susceptibility protein (CAS) bounded to Ran-GTP. Without Ran-GTP, CAS will not have high affinity for ?-Karyopherin(Kutay et al., 1997) . Cargo is now unloaded in the nucleus.CAS carrying ?-Karyopherin, and ?-karyopherin both interact with NPC to cross the nuclear membrane. This movement is driven by the Ran-GTP concentration gradient between nucleus and cytoplasm, nucleus having the higher concentration. In the cytoplasm, Ran-GAP (GTPase activating protein) binds to Ran-GTP, activating the GTPase causing hydrolysis of GTP to GDP. As Ran-GDP has a lower affinity for ?-karyopherin and CAS, it no longer binds to it (Kutay et al., 1997). Both ? and ? -Karyopherin are free to form heterodimer, proceeding to import more cargo. CAS returns into nucleus. Without Ran-GAP, Ran-GTP will remain bounded to ?-Karyopherin. Import will not occur.To return, Ran-GDP interacts with nuclear transport factor 2 (NTF2) and enters the nucleus through the NPC. Ran-GEF (guanine exchange factor) which is bonded to chromatin stimulates the dissociation of GDP in Ran-GDP so that GTP can bind to it instead (Cherfils & Chardin, 1999). With that, Ran-GTP can undock the cargo from incoming importins.