CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.