Nuclear transport of the nitric oxide synthase interacting protein (NOSIP)
by Marius Philip Pörschke
Date of Examination:2022-10-21
Date of issue:2023-05-23
Advisor:Prof. Dr. Ralph H. Kehlenbach
Referee:Prof. Dr. Ralph H. Kehlenbach
Referee:Prof. Dr. Hauke Hillen
Persistent Address:
http://dx.doi.org/10.53846/goediss-9877
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Abstract
English
Proteins that need to actively enter the nucleus are thought to be transported by a specific nuclear transport receptor (NTR), depending on the type of the nuclear localization signal (NLS). Some cargoes are reported to be transported by several NTRs, like Histones, HIV-1-Rev or FUS. In a recent proteomic screen for importin 13 cargoes, Lipin 1 and NOSIP were found as potential import cargoes. However, both cargoes had been suggested to be imported by importin α/β. In this study the role of importin 13 and other NTRs in the nuclear import of Lipin 1 and NOSIP was analyzed. Lipin 1 has a dual function as transcriptional coactivator and as phosphatidate phosphatase. For both functions, the subcellular localization is important. Lipin 1 directly interacted with importin 13 and importin α/β. Further, importin α/β was confirmed as the preferred NTR for Lipin 1. However, importin 13 could partially rescue an importin β knockdown, suggesting that importin 13 is involved in the nuclear import of Lipin 1 as well, perhaps under specific conditions or in specific cell types. Moreover, the region comprising amino acids 398-414 was found to contain a CRM1-dependent NES. The best characterized function of NOSIP is the regulation of eNOS activity by translocating the membrane bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to translocate from the nucleus to the cytoplasm. The strong nuclear accumulation of NOSIP was shown to depend on active nuclear import, whereas export depends only on passive diffusion. The cytoplasmic enrichment of NOSIP seemed to be regulated through phosphorylation. A phosphomimic mutant of NOSIP was enriched in the cytoplasm, but the nuclear transport was not affected, pointing to a retention of NOSIP through binding to a cytoplasmic binding partner. Moreover, NOSIP specifically interacted with multiple NTRs in a RanGTP-dependent manner. In competition assays, transportin 1 was able to replace all other NTRs from binding to NOSIP. In addition, knockdown experiments showed that transportin 1 is the major NTR for NOSIP. Interestingly, NOISP binds transportin 1 in an unusual binding-mode. The binding-site of NOSIP on transportin 1 was mapped to the N-terminal arch using crosslinking combined with mass spectrometry and interaction studies. This is in contrast to typical PY-NLS or RG/RGG-motif containing transportin 1 cargoes, which bind to the C-terminal arch of transportin 1. This N-terminal binding to NTRs was also observed for importin β and importin 13. No specific region or NLS like sequence of NOSIP could be identified. Instead, using different NOSIP fragments for binding assay and localization studies in cells, showed that several regions are important for the nuclear localization of NOSIP, suggesting that folded domains of NOSIP function as nuclear localization signals.
Keywords: NOSIP; Nuclear transport; Transportin 1
