Cells can shuffles molecules in and out of the nucleus through several different pathways. The one using nuclear localisation signals is outlined below.
In the heterodimeric importin, importin-alpha recognises the NLS, a motif of basic amino acids that tags a molecule as cargo. The NLS can occur as one motif (monopartite) or two motifs (bipartite). Importin-alpha contains several armadillo (ARM) repeats, which forms a curving structure with two NLS-binding sites within a shallow groove: the major and minor NLS-binding sites. The major NLS-binding site is close to the N-terminus of importin-alpha, and binds monopartite, as well as one motif of bipartite NLS cargo. The minor NLS-binding site is closer to the C-terminus of importin-alpha, and binds the second motif of bipartite NLS cargo.
In a similar way, certain exportins recognise and bind to nuclear export sequences (NES) in their RNA or protein cargo for export from the nucleus to the cytoplasm.
The rate importin can transfer cargo to the nucleus depends upon both the binding affinity of the cargo’s NLS region for importin-alpha, and the concentration of importin-alpha present.
In addition to the rate of transfer, transportation also needs to be unidirectional to be effective. The one-way transfer of cargo is in part achieved through the compartmentalisation of Ran, a Ras-type GTPase that acts to control the binding and release of cargo. Ran can exist as RanGTP and RanGDP, its form controlled in part by regulatory proteins. In the cytoplasm, the presence of the regulator Ran GTPase Activating Protein (RanGAP) helps ensure a source of RanGDP. In the nucleus, the presence of the regulator Ran Guanine Nucleotide Exchange Factor (RanGEF) helps ensure a source of RanGTP.
Importins release cargo in the presence of RanGTP, while exportins bind cargo in its presence. Therefore, the predominance of RanGTP within the nucleus and RanGDP within the cytoplasm helps to ensure that importins release their cargo within the nucleus upon RanGTP binding, and exportins release their cargo in the cytoplasm upon hydrolysis of RanGTP to GDP.
Once the importin-beta/importin-alpha/cargo trimeric complex reaches the nucleus, it must release its cargo, which is achieved in multiple ways. The primary factor is the binding of RanGTP to importin-beta, which causes a conformational change that releases importin-beta from the importin-alpha/cargo complex. Consequently, the N-terminal domain (importin-beta-binding IBB domain) of importin-alpha is freed from it binding with importin-beta. The N-terminal IBB domain of importin-alpha contains an auto-inhibitory region that mimics the NLS of its cargo. This auto-inhibitory region folds over and competes for binding with cargo at the major NLS-binding site of importin-alpha. Both monopartite and bipartite NLS cargo bind to the major NLS-binding site of importin-alpha; therefore, both types of cargo will be released upon binding of the auto-inhibitory region.
In addition, some importins make use of release factors, such as Nup2, Nup50 or Nup60, to help release cargo once it reaches the nucleus.
Furthermore, the binding of the export receptor for importin-alpha (required for recycling importin-alpha) helps release it from its cargo.