As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked ␤-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild ␤-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using ␤-elimination/Michael addition methods. The rapid identification of proteins by mass spectrometry has become commonplace in the postgenomic era (1). However, one major challenge that remains is the identification of post-translational modifications on these proteins. More than 25 years ago, Finn Wold and colleagues (2) recognized the abundance of naturally occurring modified forms of the genetically encoded 21 amino acids. In addition to phosphorylation, a variety of post-translational modifications, including acetylation (3), methylation (4), and O-linked ␤-N-acetylglucosamine (OGlcNAc) 1 (5-7), are now recognized to regulate protein functions in cellular processes. Therefore, identification of proteins along with their post-translational modifications, which has been referred to as "functional proteomics," is an important step in the characterization of proteomes. O-GlcNAc is a dynamic post-translational modification occurring on a variety of nucleocytoplasmic proteins and, in several instances, O-GlcNAc maps to the same or adjacent sites as phosphorylation (8, 9). Diverse classes of proteins are modified including cytoskeletal proteins, transcription factors, signaling adapter molecules, hormone receptors, nuclear pore complex (NPC) proteins, and kinases (10). The nucleocytoplasmic enzymes for the addition (O-GlcNAc transferase) and removal (neutral ␤-N-acetylglucosaminidase, O-GlcNAcase) of this modification have been cloned and characterized (11-16) and may ac...

Triple A syndrome is a human autosomal recessive disorder characterized by an unusual array of tissue-specific defects. Triple A syndrome arises from mutations in a WD-repeat protein of unknown function called ALADIN (also termed Adracalin or AAAS). We showed previously that ALADIN localizes to nuclear pore complexes (NPCs), large multiprotein assemblies that are the sole sites of nucleocytoplasmic transport. Here, we present evidence indicating that NPC targeting is essential for the function of ALADIN. Characterization of mutant ALADIN proteins from triple A patients revealed a striking effect of these mutations on NPC targeting. A variety of disease-associated missense, nonsense, and frameshift mutations failed to localize to NPCs and were found predominantly in the cytoplasm. Microscopic analysis of cells from a triple A patient revealed no morphological abnormalities of the nuclei, nuclear envelopes, or NPCs. Importantly, these findings indicate that defects in NPC function, rather than structure, give rise to triple A syndrome. We propose that ALADIN plays a cell type-specific role in regulating nucleocytoplasmic transport and that this function is essential for the proper maintenance and͞or development of certain tissues. Our findings provide a foundation for understanding the molecular basis of triple A syndrome and may lead to unique insights into the role of nucleocytoplasmic transport in adrenal function and neurodevelopment.

The Mre11/Rad50/NBS1 (MRN) protein complex and ATMIN protein mediate ATM kinase signaling in response to ionizing radiation (IR) and chromatin changes, respectively. NBS1 and ATMIN directly compete for ATM binding, but the molecular mechanism favoring either NBS1 or ATMIN in response to specific stimuli is enigmatic. Here, we identify the E3 ubiquitin ligase UBR5 as a key component of ATM activation in response to IR. UBR5 interacts with ATMIN and catalyzes ubiquitination of ATMIN at lysine 238 in an IR-stimulated manner, which decreases ATMIN interaction with ATM and promotes MRN-mediated signaling. We show that UBR5 deficiency, or mutation of ATMIN lysine 238, prevents ATMIN dissociation from ATM and inhibits ATM and NBS1 foci formation after IR, thereby impairing checkpoint activation and increasing radiosensitivity. Thus, UBR5-mediated ATMIN ubiquitination is a vital event for ATM pathway selection and activation in response to DNA damage.A TM kinase is part of the phosphatidylinositol 3-kinaserelated kinase (PIKK) family that activates cell-cycle checkpoints and promotes DNA repair in response to DNA damage or replication blocks (1). Mutation of ATM causes the genomic instability syndrome ataxia telangiectasia, characterized by cerebellar degeneration, immunodeficiency, and increased tumor incidence (2).In response to DNA double-strand breaks (DSBs), inactive ATM homodimers dissociate and the kinase is activated (3), phosphorylating other ATM molecules, as well as numerous substrates including structural maintenance of chromosomes protein 1 (SMC1) and p53, at serine or threonine residues followed by glutamine (the "SQ/TQ" motif) (4, 5). ATM is activated at DSB sites via the Mre11/Rad50/NBS1 (MRN) complex, which is required for ATM activation and recruitment into nuclear foci, and MRN interacts with ATM mainly via its NBS1 subunit (6, 7). A short C-terminal motif in NBS1 principally contributes to ATM binding (8), and the interaction is also strengthened by ubiquitination of NBS1 (9).ATM not only is central to the DSB response but also responds to many other cellular stresses, such as UV damage and hypotonic stress (1, 3). In contrast to its role at DSB sites, NBS1 is not required for ATM activation by these stimuli (10, 11); instead, ATM is activated via interaction with its cofactor ATMIN (12). Accordingly, ATMIN colocalizes with phosphorylated ATM in basal conditions and after hypotonic stress, but not after ionizing radiation (IR). The mechanism of the switch between these different signaling conditions is incompletely understood. Our previous work has indicated that competitive interaction of either NBS1 or ATMIN with ATM is part of this switch and that overexpression of ATMIN can inhibit NBS1-mediated ATM activation (11). However, the signaling mechanism(s) that favor interaction with one protein over the other in different conditions are unknown.Ubiquitin protein ligase E3 component n-recognin 5 (UBR5) is a very large protein of 2,799 amino acids (309 kDa) belonging to the HECT family of E3 ubiquitin ...