Nucleo cytoplasmic trafficking
a. Recognition of Cargo Proteins
Due to the fact that the nucleus is degraded and rebuilt every cell cycle, proteins with prolonged half life have to be imported in the nucleus after every re-formation of the nuclear envelope. Others have to shuttle between the nuclear and cytoplasmic compartment to fulfill their duty. Thus the recognition requires a permanent surface exposed signal which is not cleaved off after translocation like for other transport pathways e.g. in the ER and thus may be an integral part the protein. This is a general feature of proteins trafficking the nuclear envelope.
The first localization signal identified for the import and export pathway have been named classical NLS (cNLS, nuclear localisation signal) and NES (nuclear export signal), repectively (Fig. 1). They are recognized by CRM1 for NESs or an importin-ß-dependent pathway for the classical NLSs. The interaction of NLS bearing cargoes with Importin-ß is indirect as it requires a bridging or „adaptor" molecule, for example Importin-α.
Subsequently, further general import signals have been identified, like the PY-motif (Fig. 1) for cargoes imported by Transportin, another member of the Importin-ß superfamily, which directly interacts with the cargo.
Fig1. Nuclear Transport signals.
The classical NLS may be monopartite - with 5 out of 7 residues basic - or bipartite. The second part is 10-12 residues distant and composed of a short stretch of basic residues named basic patch (BP). The classical NES in contrast exhibits a series of hydrophobic residues separated by 1-3 residues.
The variability of cargoes has led to a huge variety of localisation signals. In order to fulfill the requirements to achive the recognition of all the different signals, karyopherins have evolved the mentioned flexibility and often binding regions with a broad albeit specific recognition capability (Fig.2, left). Moreover they may contain more than one binding region or use adaptors to bind cargoes.
Fig2. NLS-cargo import receptor interaction scenarios.
Importin-ß like Transportin may bind cargo directly as indicated for Importin-ß in the left panel. The blue circle highlights the helices of the Sterol-receptor binding protein foung in the crystal structure. Importin-ß is the most versatile receptor, as it also may bind cargoes via an adaptor molecule like Impa or SPN1. Moreover cargo binding requiring the interaction with a second receptor has been found.
Fig3. Model of the Importin-ß/Importin-α heterodimer.
Importin-ß binds the IBB-domain of Importin-α(blue helix) and a short linker connects to the cargo binding domain.
Adaptors themselves bear regions mediating specific binding to the receptor and a separate specific binding region for the cargo, thus bridging the interaction between cargo and receptor, increasing the number of localisation signals even more (Fig. 2, middle). Importin-ß is such a receptor that has multiple ways of recognising its cargoes. Either it recognizes the cargo directly or uses the help of such adaptor molecules to achieve its task. Two adaptors, Importin-α and Snurportin 1 (SPN1), have been extensively studied (Figs. 3 and 4). Besides the diverging cargo binding regions - one binds proteins, the other specific cap structures of UsnRNAs (Read More). Both share a similar N-terminal region for Importin-ß binding, the IBB domain (Fig. 4). Interestingly, both adaptors require exportins for their recycling to the cytoplasm. Whereas SPN1 is exported by a general export receptor CRM1 (Chromosome region maintenace 1; also making use of adaoptors to achieve its task), Importin-αis exported by a specific exportin, CAS (Cellular Apoptosis Susceptibility gene product).
IBB-domain structure
The N-terminal IBB-domain of Snurportin1 binds to the C-terminal region of Importin-ß similar to the IBB domain (Fig.4) of a second well studied adaptor Importin-α surprisingly differences in the IBB domain sequence result in differences in the disassembly of the import complex. Whereas RanGTP binding to Importin-ß bound to Importin-α requires RanGTP for release from the NPC and disassembly, Importin-ß bound to SPN1 requires RanGTP only fort the latter.
Fig.4. IBB-domain of SPN1 bound to Importin ß.
The crystal structure of the IBB-domain from SPN1 bound to Importin-ß exhibits an extended conformation similar to the one observed in Fig 3 (purple helix) in contrast to the Importin-ß bound to the IBB-domain of Importin-α.
References
J. Mol. Biol. 374, 1129-1138. [Abstract]; PDB:[2QNA]
Further reading