The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.

Short intense bouts of MERE can trigger increases in circulating EPC and related angiogenic factors, potentially contributing to vascular adaptation and vasculoprotection.

Nitric oxide synthase is inhibited by asymmetric NG-methylated derivatives of arginine whose cellular levels are controlled in part by dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18). Levels of asymmetric NG,NG-dimethylarginine (ADMA) are known to correlate with certain disease states. Here, the first structure of a DDAH shows an unexpected similarity to arginine:glycine amidinotransferase (EC 2.1.4.1) and arginine deiminase (EC 3.5.3.6), thus defining a superfamily of arginine-modifying enzymes. The identification of a Cys-His-Glu catalytic triad and the structures of a Cys to Ser point mutant bound to both substrate and product suggest a reaction mechanism. Comparison of the ADMA-DDAH and arginine-amidinotransferase complexes reveals a dramatic rotation of the substrate that effectively maintains the orientation of the scissile bond of the substrate with respect to the catalytic residues. The DDAH structure will form a basis for the rational design of selective inhibitors, which are of potential use in modulating NO synthase activity in pathological settings.

Adrenal hypoplasia congenita (AHC) causes primary adrenal insufficiency due to the failure of development of the adrenal cortex. Clinical and pedigree data indicate that the condition is genetically heterogeneous. The predominant adrenal hypoplasia congenita locus, however, is the NR0B1 gene, at Xp21, encoding the protein DAX1. In this article, we present a compendium of published NR0B1 mutations and polymorphisms, and discuss them in the contexts of known biology and clinical applicability. The recent descriptions of patients with primary adrenal insufficiency due to mutations of NR5A1, which encodes SF1, are also discussed.

The ␥-aminobutyric acid receptor type A (GABA A ) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA A receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 Å resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the ␥2 subunit of the GABA A receptor and the acidic carboxylterminal tails of human ␣-and ␤-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.The ␥-aminobutyric acid receptor type A (GABA A ) 1 is a ligand-gated chloride ion channel that mediates inhibitory neurotransmission (1). The ␥-aminobutyric acid receptor type Aassociated protein (GABARAP) was identified as interacting with the M3-M4 loop of the ␥2 subunit of the GABA A receptor by yeast two-hybrid analysis and co-immunoprecipitation (2). The initial two-hybrid clone identified consisted of the carboxyl-terminal portion (residues 36 -117) of the complete 117-residue GABARAP protein. A distant homology to microtubuleassociated protein 1A and microtubule-associated protein 1A and 1B light chain 3 (MAP-LC3) (3) identified GABARAP as a possible tubulin-binding protein. GABARAP was found to be associated with microtubules (2, 4), and the highly basic aminoterminal region of the GABARAP protein was putatively identified as the tubulin binding region. Thus, it has been suggested that GABARAP serves to link the GABA A receptor (through interactions with the carboxyl-terminal 35-117 region) to the cytoskeleton (via the amino-terminal region) and is involved in organizing GABA A receptors at the synapse (2, 5). Other experiments have not supported location of GABARAP at the synapse (6) but instead indicate interaction with gephyrin, a protein which itself binds tubulin and the glycine receptor (7) and has a role in the postsynaptic localization of GABA A receptors.Sequence analysis (4, 6) identified a number of orthologues of GABARAP. In addition to MAP-LC3, these included yeast autophagy protein Aut7p (sequences from three species exist) and a protein involved in intra-Golgi transport (GATE-16), also known as ganglioside expression factor 2. More recently, an additional homologue has been identified as an early estrogenregulated gene, gec1 (8). Three GABARAP homologues have been identified from cDNA data base searching (9); two are ...

We provide evidence from comparisons of populations of Drosophila that evolutionary correlations between longevity and stress resistance break down over the course of laboratory evolution. Using 15 distinct evolutionary regimes, we created 75 populations that were differentiated for early fecundity, longevity, starvation resistance, desiccation resistance, and developmental time. In earlier experiments, selection for postponed aging produced increases in stress resistance, whereas selection for increased stress resistance produced increases in longevity. Direct estimates of correlations also indicated an antagonistic relationship between early fecundity on one hand and longevity or stress resistance on the other. Laboratory evolution of extreme values of stress resistance, however, led to a breakdown in these evolutionary relationships. There was no evidence that these significant changes in correlation resulted from genotype-by-environment interactions or inbreeding. These findings suggest that correlations between functional characters are not necessarily durable features of a species, and that short-term evolutionary responses cannot be extrapolated reliably to longer-term evolutionary patterns.

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Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction‐resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site‐directed mutagenesis identifies residues involved in catalysis.