3.8.1. Nuclear targeting :
Proteins are not transported through the nuclear membrane but rather through a complex pore called the nuclear pore, which is comprised of-
- About 100 different proteins
- Proteins smaller than 20 KDa by selective transport
- Proteins larger than 20KDa by selective transport (nuclear localization signal; NLS), which is a cluster of 4-8 positively charged amino acids (eg- PKKKRLV)
Usually, nucleus targeted proteins follows two way traffic: in and out
In: Nucleoplasm involves proteins, DNA.
Inside nucleus, DNA and RNA polymerases, transcription factors, histones etc. are targeted across the nuclear pore.
Out: Outside nucleus, mRNA, tRNA, rRNA are generally targeted across the nuclear pore.
Proteins are targeted to the nucleus by a specific amino acids sequence as phenylalanine glycine repeats (FG repeats) while some proteins exits from nuclear requires a nuclear export sequences (NES). Nuclear import & export pathways are mediated by a family of soluble receptor referred to as importin and exportin and collectively called karyopherins (alpha/importin beta1 heterodimer; designated as a and b). The best studied NLS are basic amino acids sequence typically rich in Lys and Arg.
The best characterized nuclear transport sequence are the small hydrophobic leucine rich nuclear export sequences, 1st described in HIV Rev protein ( Crm-1 exportin).An importin binds to its NLS nearing cargo/protein in the cytoplasm and translocates through the NPC into the nucleus. The importin binds Ran–GTP in the nucleus, resulting in cargo varies. The importin–Ran–GTP complex recycles back into the cytosol after translocation to the cytoplasm, GTP hydrolysis on Ran by Ran–GAP.
A gradient of Ran–GTP exists in the cell with Ran–GDP add high concentration in the cytosol and Ran–GTP at a high concentration in the nucleus to maintain the high nuclear concentration of Ran, a dedicated transporter protein NTF2 functions to recycle Ran–GDP continuously back to the nucleus. Ran–GTP is generated in the nucleus by chromatin bound Ran–GEF.
Cargo protein (protein which needs to transport from cytoplasm to nucleus) for importin present in cytoplasm. Impotin get responsible to transfer it respective cargo from cytoplasm to nucleus, to accomplish this function binding between cargo and importin takes place, in turn they become capable to make interaction with nuclear pore and as a result pass from its cannel and comes inside nucleus. In nucleus Ran–GTP complex binds to importin and cargo complex which cause conformational change in importin, in turn, importin dissociation with its cargo takes place, as a result cargo get transported to cytoplasm and now translocation of Ran–GTP complex along with importin from the nucleus to the cytoplasm takes place. In cytoplasm Ran binding protein (Ran BP) binds to Ran and cause the separation of Ran–GTP complex from importin, as a result a access generate for GTPase activating protein (GAP) to get bind to Ran–GTP complex, in turn cause hydrolysis of GTP occur and formation of Ran–GDP takes place. Ran–GDP binds to nuclear transport factor which mediate its transfer from cytoplasm to nucleus. In nucleus Ran–GDP complex trigger by guanine nucleotide exchange factor which convert GDP into GTP and formation of Ran–GTP complex takes place, now this complex again start new cycle.
In nucleus exportin binds to its respective cargo protein, then this complex binds with Ran–GTP complex and along with Ran–GTP complex, exportin and its cargo get diffuse from the nuclear pore and reach into the cytoplasm. In cytoplasm GAP protein binds to Ran–GTP complex and causes hydrolysis of GTP in turn cargo get release in nucleus. After that Ran–GDP move into the nucleus with a ligand which it takes from cytoplasm and in nucleus it again get converted in to Ran–GTP by the action of GEF and release is ligand in nucleus.