The venom enzyme sphingomyelinase D (SMase D) in the spider family Sicariidae (brown or fiddleback spiders [Loxosceles] and six-eyed sand spiders [Sicarius]) causes dermonecrosis in mammals. SMase D is in a gene family with multiple venom-expressed members that vary in functional specificity. We analyze molecular evolution of this family and variation in SMase D activity among crude venoms using a data set that represents the phylogenetic breadth of Loxosceles and Sicarius. We isolated a total of 190 nonredundant nucleotide sequences encoding 168 nonredundant amino acid sequences of SMase D homologs from 21 species. Bayesian phylogenies support two major clades that we name alpha and beta, within which we define seven and three subclades, respectively. Sequences in the alpha clade are exclusively from New World Loxosceles and Loxosceles rufescens and include published genes for which expression products have SMase D and dermonecrotic activity. The beta clade includes paralogs from New World Loxosceles that have no, or reduced, SMase D and no dermonecrotic activity and also paralogs from Sicarius and African Loxosceles of unknown activity. Gene duplications are frequent, consistent with a birth-and-death model, and there is evidence of purifying selection with episodic positive directional selection. Despite having venom-expressed SMase D homologs, venoms from New World Sicarius have reduced, or no, detectable SMase D activity, and Loxosceles in the Southern African spinulosa group have low SMase D activity. Sequence conservation mapping shows >98% conservation of proposed catalytic residues of the active site and around a plug motif at the opposite end of the TIM barrel, but alpha and beta clades differ in conservation of key residues surrounding the apparent substrate binding pocket. Based on these combined results, we propose an inclusive nomenclature for the gene family, renaming it SicTox, and discuss emerging patterns of functional diversification.

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Dltv1 mutants, as judged by the altered distribution of the 59-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.T HE synthesis of ribosomes in eukaryotes is a complex and evolutionarily conserved process that involves the processing and folding of four rRNAs and the sequential incorporation of .70 ribosomal proteins into two subunits. Ribosome biogenesis has been studied most extensively in the yeast Saccharomyces cerevisiae, where genetic and biochemical studies have identified .170 nonribosomal factors that are required for, or are members of, large complexes that form as intermediates in the assembly process (for review, see Verma et al. 1995;Kressler et al. 1999; FromontRacine et al. 2003;Tschochner and Hurt 2003;Granneman and Baserga 2004;Dlakic 2005). These trans-acting factors include RNA exo-and endonucleases, RNA-modification enzymes, helicases, and export factors, as well as a large number of proteins whose functions are as yet unknown. Current models of ribosome assembly suggest that a number of factors important in 40S biogenesis, as well as a subset of early assembling ribosomal proteins, initially assemble cotranscriptionally on the 35S pre-rRNA to form a 90S preribosomal complex. Cleavage of the 35S rRNA in the 90S complex at sites A 0 , A 1 , and A 2 , which requires the U3 small nucleolar RNA, separates the pre-40S precursor from the pre-60S. Most of the accessory factors are released from the pre-40S at this point, which rapidly exits the nucleus in the company of a much smaller number of new factors necessary for export. Final maturation of the 40S subunit, which includes cleavage of the 20S rRNA to yield the mature 18S rRNA, occurs in the cytoplasm. The 60S-processing machinery is recruited after the release of the 40S precursor. The pre-60S subunit undergoes a series of RNA-processing reactions in the nucleolus before being released to the nucleoplasm w...

Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through divergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.

In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of ⌬yar1 mutants, but does not suppress either defect in ⌬ltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stresssensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

We have found a novel modification of protein arginine residues in the yeast Saccharomyces cerevisiae. Intact yeast cells lacking RMT1, the gene encoding the protein -N G -arginine methyltransferase, were labeled with the methyl donor S-adenosyl-L-[methyl-3 H]methionine. The protein fraction was acid-hydrolyzed to free amino acids, which were then fractionated on a high resolution sulfonated polystyrene cation exchange column at pH 5.27 and 55°C. In the absence of the -N G ,N G -[ 3 H]dimethylarginine product of the RMT1 methyltransferase, we were able to detect a previously obscured 3 H-methylated species that migrated in the region of methylated arginine derivatives. The [ 3 H]methyl group(s) of this unknown species were not volatilized by treatment with 2 M NaOH at 55°C for up to 48 h, suggesting that they were not modifications of the terminal -guanidino nitrogen atoms. However, this base treatment did result in the formation of a new 3 H-methylated derivative that co-chromatographed with ␦-N-methylornithine on high resolution cation exchange chromatography, on reverse phase high pressure liquid chromatography, and on thin layer chromatography. From these data, we suggest that the identity of the original unknown methylated residue is ␦-N-monomethylarginine. The presence of this methylated residue in yeast cells defines a novel type of protein modification reaction in eukaryotes.