ABSTRACT

؉ RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

Y.Strahm and B.Fahrenkrog contributed equally to this workGle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets. In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/ Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X n ‫؍‬ 3-7 )-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II␣. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003376, 1-15, 2011. Phosphoinositides (PIs)1 are lipid second messengers unique among phospholipids: Their inositol head group is rapidly phosphorylated by specific lipid kinases yielding seven distinct biologically relevant phosphatidylinositol derivatives. The coordinated activities of the PI-specific kinases and phosphatases generate PI profiles, which contribute to downstream signaling events regulating a variety of cellular processes such as proliferation, cell survival, migration, and vesicular trafficking (1-4). Impairment of PI metabolism is associated with cancer as well as neurological and immunological disorders (5-7). PIs are not only substrates for the generation of second messengers but are also second messengers themselves. They have emerged as sensors for specific PI-binding domains present in a diverse array of proteins: PH (pleckstrin homology), epsin N-terminal homology, FYVE (Fab1, YOTB, Vac1, EEA1), Phox homology, PHD (plant homeodomain), PDZ domains as well as unstructured lysine/ arginine-rich patches. These domains display a range of heterogeneity in terms of their specificity for the different PIs (8 -10) and recruit target, domain-containing, effector proteins in a temporal and spatial manner to sites of PI synthesis at many cytoplasmic locations (11).PIs (notably phosphatidylinositol(3)phosphate (PtdIns(3)P), PtdIns(4)P, PtdIns(5)P, PtdIns(4,5)P 2 , PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 ) have also been iden...

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX 5 P in Imp1 and NX 5 S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. INTRODUCTIONProgrammed cell death is a means whereby metazoans can remove unwanted cells, with failure of programmed cell death enabling cancer and autoimmune disease, and inappropriate cell death contributing to neurodegenerative disease. Several of the proteins that regulate cell death, including Bcl-2 family members, signal-transduction receptors, effector proteases and DIABLO/Smac, are specifically enclosed within subcellular structures allowing precise control over the commitment of a cell to die. Understanding where and how these key regulators are localized is crucial in understanding their normal biological function and their role in the pathogenesis of malignant diseases.A family of intracellular proteases called caspases implement programmed cell death (Ekert et al., 1999). The activity of caspases is regulated by a family of inhibitor of apoptosis proteins (IAPs) that bind and neutralize active caspases (Deveraux and Reed, 1999). For example, the inhibitor MIHA/XIAP/hILP/BIRC4 can bind and inhibit processed caspases 3, 7, and 9 (Duckett et al., 1996;Liston et al., 1996;Uren et al., 1996;Deveraux et al., 1997Deveraux et al., , 1998. This damping of caspase activity provides a layer of regulation over the cell death-promoting activities of this family of effector proteases.In mammalian cells, signals for cell death can lead to rupture of the mitochondrial outer membrane, and under these conditions the inhibition of caspases can be antagonized by the mitochondrial proteins DIABLO/Smac Verhagen et al., 2000) and HtrA2/Omi (Suzuki et al., 2001;Martins et al., 2002;Verhagen et al., 2002). Structural studies Liu et al., 2000;Wu et al., 2000) have shown that purified DIABLO is a homodimer , and each DIABLO dimer can bind to the BIR domains of inhibitor proteins via contacts made to the N-terminal residues of DIABLO (Liu et al., 2000;Wu et al., 2000). The avid interaction DIABLO makes with the inhibitor MIHA competes the inhibitor away from active caspase 9, freeing caspase 9 to proteolytically activate downstream caspases .Mouse DIABLO is translated as a 237-residue precursor protein (preDIABLO) with an N-terminal presequence that must be cleaved to generate the mature form with the amino-ter...