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Merck

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein.

The Journal of biological chemistry (2023-01-24)
Marius Pörschke, Inés Rodríguez-González, Iwan Parfentev, Henning Urlaub, Ralph H Kehlenbach
RESUMEN

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase 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 be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-β, -7, -β/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.

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Sigma-Aldrich
Cicloheximida, from microbial, ≥94% (TLC)
Sigma-Aldrich
Anti-HA antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-Transportin 1 antibody, Mouse monoclonal, clone D45, purified from hybridoma cell culture
Sigma-Aldrich
Anti-NOSIP antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody