RanBPM/RanBP9 is a ubiquitous nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. effect on RanBPM localization and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally in the nucleus a significant fraction of RanBPM is associated with chromatin. Altogether these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly likely through interactions with other proteins and by intramolecular folding. Introduction Transport in and out of the nucleus of proteins above 50KDa is an active process that requires the nucleocytoplasmic transport machinery . Import to the nucleus is mediated by a nuclear localization signal (NLS) that is recognized by an import receptor (importin) which transports its cargo through the nuclear membrane in an energy-dependent process . Conversely nuclear export is dependent on a nuclear export sequence (NES) that is recognized by exportins which transport the protein out of the nucleus. Nuclear localization TBLR1 sequences fall into three classes: a short stretch of basic amino acids a bipartite NLS consisting of two short stretches of basic residues separated by 10-12 amino acids and a combination of charged/polar and non-polar residues flanked by proline and aspartic acid residues [3 4 The most common characterized NES consists of a non-conserved motif made up of hydrophobic residues and is leucine-rich . Nucleocytoplasmic transport is a tightly monitored process regulated at many different stages [2 3 One mechanism of regulation includes importin protein expression as different importins recognize different Adapalene cargoes. Another mechanism of regulation involves alteration of sequence affinity to karyopherins for example by phosphorylation of the signal sequence. A third mechanism of regulation involves intermolecular or intramolecular masking of signal sequences. This occurs through protein-protein interactions and conformational changes respectively which prevent signal recognition by karyopherins [2 3 In addition nonconventional mechanisms exist which do not rely on importins/karyopherins but on interaction with other transporters or through direct binding to nuclear pore complex components . Ran binding protein M (RanBPM also referred to as RanBP9) is a ubiquitous Adapalene nucleocytoplasmic 90kDa protein whose function is poorly understood. RanBPM contains three conserved domains (Fig. 1A) none of which confers enzymatic activity or is indicative of any specific function apart from protein interactions. The SPRY (SplA and Ryanodine receptor) domain is a protein interaction domain present in protein Adapalene families regulating a wide range of functions including regulation of cytokine signaling RNA metabolism and protein degradation . The LisH/CTLH (LIS1-homology motif/C-terminal to LisH) domain is found in proteins associated with microtubule dynamics cell migration and chromosome segregation and mediates dimerization [7-9]. The CRA (CT11-RanBPM) domain Adapalene is an α-helical structure of unknown function but is structurally reminiscent of the death domain superfamily . Fig 1 Deletion of RanBPM C-terminus does not alter its subcellular localization. RanBPM has been shown to interact with numerous proteins implicating it in a variety of cellular processes including cell adhesion migration microtubule dynamics and gene transcription [11-19]. It has been hypothesized that RanBPM functions as a scaffolding protein that may Adapalene be part of a large complex [20-22]. RanBPM has been identified as a phosphoprotein and its phosphorylation is increased in response to stress stimuli such as osmotic shock ultraviolet light (UV) and ionizing radiation (IR).