NSF/Bio-DFG: Structural and functional characterization of NOSIP, an unusual cargo of the nuclear import receptor transportin

About This Grant

All proteins are made in the cytoplasm, but thousands must enter the nucleus. The transport of these proteins from the cytoplasm to the nucleus is thus critical for proper functions of the cell. For many such proteins, nuclear localization signals (NLS) have been identified that mediate the interaction with a nuclear import receptor, which, in turn, facilitates translocation of the transport complex across the nuclear membrane. Nuclear importation is an important aspect of cell signaling. In this study a protein involved in nitric oxide signaling will be studied in order to gain mechanistic insight into how cytoplasmic signals are relayed to the nucleus. This protein (nitric oxide synthase; NOSIP) is a cargo for the prominent nuclear import receptor Transportin (TNPO1) and molecular details of this interaction will be examined. The project will involve complimentary expertise of the Kehlenbach and Chook laboratories. The cellular, biochemical and structural research project will directly link to a Broader Impact project which provides STEM training to local community college students, through hands-on lab work, literature review, generation of public educational resources and development of a comprehensive database of nuclear import cargoes. The project will help prepare the STEM workforce, educate the public and update the research community about nuclear transport, and also provide the research community with updated bioinformatics resources. Two major research questions will be addressed in the study of atypical recognition of NOSIP by TNPO1. The first investigates the mechanism of NOSIP recognition by TNPO1, with the goal of deciphering the distinct and novel interaction mode. The approach will include the Chook group solving the cryo-electron microscopy structure of the TNPO1-NOSIP complex and comparing it with those of “classic” TNPO1-PY-NLS structures. The Chook group will also compare the binding of NOSIP to TNPO1 with the interactions of NOSIP with its minor nuclear import receptors. The second major research questions will involve the Kehlenbach group investigating how the interaction of NOSIP with transport receptors and its subcellular localization are regulated by phosphorylation, and how this post-translational modification affects possible nuclear functions such as mRNA-splicing. Together, the Chook and Kehlenbach groups will 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 role in the nucleus. This collaborative US/Germany project is supported by the US National Science Foundation and the German Deutsche Forschung Gemeinschaft (DFG) where NSF funds the US investigator and DFG funds the German partner. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.