Purification of nuclear localization signal-containing proteins and its application to investigation of the mechanisms of the cell division cycle.

The GTP bound form of the Ran GTPase (RanGTP) in the nucleus promotes nuclear import of the proteins bearing nuclear localization signals (NLS). When nuclear envelopes break down during mitosis, RanGTP is locally produced around chromosomes and drives the assembly of the spindle early in mitosis and the nuclear envelope (NE) later. RanGTP binds to the heterodimeric nuclear transport receptor importin α/β and releases NLS proteins from the receptor. Liberated NLS proteins around chromosomes have been shown to play distinct, essential roles in spindle and NE assembly. Here we provide a highly specific protocol to purify NLS proteins from crude cell lysates. The pure NLS fraction is an excellent resource to investigate the NLS protein function and identify new mitotic regulators, uncovering fundamental mechanisms of the cell division cycle. It takes 2-3 days to obtain the NLS fraction.