Discovery of Nuclear Localization Signals
The presence of the nuclear membrane that sequesters the cellular DNA is the defining feature of eukaryotic cells. The nuclear membrane therefore separates the nuclear processes of DNA replication and RNA transcription from the cytoplasmic process of protein production. Proteins required in the nucleus must be directed there by some mechanism. The first direct experimental examination of the ability of nuclear proteins to accumulate in the nucleus were carried out by John Gurdon when he showed that purified nuclear proteins accumulate in the nucleus of frog (Xenopus) oocytes after being micro-injected into the cytoplasm. These experiments were part of a series which subsequently led to studies of nuclear reprogramming, directly relevant to stem cell research.
The presence of several million pore complexes in the oocyte nuclear membrane and the fact that they appeared to admit many different molecules (insulin, bovine serum albumin, gold nanoparticles) led to the view that the pores are open channels and nuclear proteins freely enter the nucleus through the pore and must accumulate by binding to DNA or some other nuclear component. In other words there was thought to be no specific transport mechanism.
This view was shown to be incorrect by Dingwall and Laskey in 1982. Using a protein called Nucleoplasmin, the archetypal ‘molecular chaperone’, they identified a domain in the protein which acted as a signal for nuclear entry. This work stimulated research in the area and two years later the first NLS was identified in SV40 Large T-antigen (or SV40, for short). However a functional NLS could not be identified in another nuclear protein simply on the basis of similarity to the SV40 NLS. In fact only a small percentage of cellular (non-viral) nuclear proteins contained a sequence similar to the SV40 NLS. A detailed examination of Nucleoplasmin identified a sequence with two elements made up of basic amino acids separated by a spacer arm. One of these elements was similar to the SV40 NLS but was not able to direct a protein to the cell nucleus when attached to a non-nuclear reporter protein. Both elements are required. This kind of NLS has become known as a bipartite classical NLS. The bipartite NLS is now known to represent the major class of NLS found in cellular nuclear proteins and structural analysis has revealed how the signal is recognized by a receptor (importin α) protein (the structural basis of some monopartite NLSs is also known). Many of the molecular details of nuclear protein import are now known. This was made possible by the demonstration that nuclear protein import is a two step process; the nuclear protein binds to the nuclear pore complex in a process which does not require energy. This is followed by an energy dependent translocation of the nuclear protein through the channel of the pore complex. By establishing the presence of two distinct steps in the process the possibility of identifying the factors involved was established and led on to the identification of the importin family of NLS receptors and the GTPase Ran.
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