Question

Why is it necessary for transformed plasmids to have a nuclear localization signal in order for them to go into the yeast nucleus?

Answer 1

Nuclear Localization Signal(NLS) is a sequence of amino acid which tags a protein for import into cell nucleus by nuclear transport. This signal consists of short sequences of positively charged lysines or arginines on the protein surface. Presence of nuclear membrane that sequesters cellular DNA is an important feature of eukaryotic cells( like yeast cells).Nuclear membrane separates the nuclear processes of DNA replication and transcription of RNA from cytoplasmic process of protein production. Proteins required in nucleus must be directed by some other mechanism.This lead to the discovery of NLS.

Protein translated with NLS will bind strongly to importin, and the complex will move through nuclear pore. Then Ran-GTP will bind to the importin-protein complex, this binding causes importin to lose affinity for the protein. The protein is thus released the Ran-GTP/importin complex will move back out of the nucleus through the nuclear pore.

Plasmids enter the nucleus either upon the mitotic disassembly of the nuclear envelope or through nuclear pore complexes in absence of cell division, using a different set of proteins(NLS mechanism).While DNA is released into cytoplasm it must traffic to nucleus for gene expression to occur. This translocation represents a significant barrier to gene delivery. First, cytoplasm contains nucleases that will degrade free DNA.It was found that plasmid DNA is degraded in cytoplasm with a half-life of 50–90 min. This creates problem for delivery of naked DNA and DNA–carrier complexes to nuclear entry. ‘naked DNA’ may not describe the state of DNA once released into the cytoplasm. Rather, 'naked’ DNA is complexed with a number of host cellular proteins to form protein–DNA complex that mediates subsequent interactions with intercellular pathways, condenses the plasmid to reduce its effective size and shields the DNA from rapid degradation.

In Yeast ,nuclear membrane remain intact throught the cell cycle.Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus.Ty1 generates more copies of itself for insertion into genome of host cell. Ty1 mRNA is transcribed and processed in the nucleus and then it is transported to the cytoplasm, where it is translated into Gag and Gag-Pol proteins. The transformation into nucleus is by docking of Ty1 VLPs at a nuclear pore, coupled with transfer of preintegration complex , requiring the NLS, and specific VLP disassembly process that releases complex competent for subsequent nuclear uptake .IN is a Ty1 protein that must enter into the nucleus since its insertion of Ty1 cDNA copy into chromosomal DNA,which is absolutely required for retrotransposition. Ty1 IN indeed has a bipartite nuclear localization signal (NLS) that is essential for retrotransposition.