Structural and functional characterization of NOSIP, an unusual cargo of the nuclear import receptor transportin

• Applicant: Professor Dr. Ralph Kehlenbach
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 544558352

Import of cargo proteins into the nucleus is key to cellular function and thousands of proteins are known to enter the nucleus at some point of the cell cycle. For many such proteins, nuclear localization signals (NLSs) have been identified that mediate the interaction with a nuclear transport receptor, which, in turn, facilitates translocation of the transport complex across the nuclear pore complex. One of the best-described transport receptors is transportin (TNPO1), also known as karyopherin 2, which typically interacts with a specific NLS, the proline-tyrosine- or PY-NLS. Structures of TNPO1 in complex with PY-NLS-containing proteins (or protein fragments) have been solved using X-ray crystallography, revealing a distinct NLS-binding site in the C-terminal half of the transport receptor. NOSIP (nitric oxide synthase interacting protein) has been described as a protein that affects the activity of a cytoplasmic enzyme, nitric oxide synthase. In most stages of the cell cycle, however, NOSIP localizes to the nucleus, where it was shown very recently to participate in alternative RNA-splicing. Accordingly, we could recently show that TNPO1 is a major nuclear import receptor for NOSIP. Strikingly, our data also showed that the interaction of NOSIP with TNPO1 is very different from that of the prototypic, PY-NLS-containing cargo proteins. Instead of a PY-NLS, NOSIP contains a basic stretch of amino acid residues within an extended alpha-helix, which is necessary, but not sufficient for TNPO1 binding. Furthermore, this basic stretch rather interacts with the N-terminal half of TNPO1, unlike the PY-NLS. Two major questions are addressed in this proposal: first, we plan to investigate the mechanism of NOSIP recognition by TNPO1. We will decipher this distinct and novel interaction mode by solving the structure of the TNPO1-NOSIP complex using cryo-electron microscopy and compare it with that of “classic” TNPO1-PY-NLS structures. We will also compare the binding of NOSIP to TNPO1 with its interactions with its minor nuclear import receptors. Second, we will investigate how the interaction of NOSIP with transport receptors and its subcellular localization is regulated by phosphorylation and how this affects possible nuclear functions, e.g. in RNA-splicing. Together, the project should shed light on the highly unusual interaction mode of TNPO1 with NOSIP as a cargo protein and on the regulation of the subcellular localization of NOSIP, which seems to play a prominent and unexpected role in the nucleus.

• DFG Programme: Research Grants
• International Connection: USA
• Partner Organisation: National Science Foundation (NSF)
• Cooperation Partner: Professorin Yuh Min Chook, Ph.D.