TORC2 signaling is antagonized by protein phosphatase 2A and the Far complex in Saccharomyces cerevisiae

The target of rapamycin (TOR) kinase, a central regulator of eukaryotic cell growth, exists in two essential, yet distinct, TOR kinase complexes in the budding yeast Saccharomyces cerevisiae: rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell viability. However, it is unclear whether the essential function of Lst8 is linked to TORC1, TORC2, or both. To that end, we carried out a genetic screen to isolate lst8 deletion suppressor mutants. Here we report that mutations in SAC7 and FAR11 suppress lethality of lst8Δ and TORC2-deficient (tor2-21) mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. More importantly, characterization of lst8Δ bypass mutants reveals a role for protein phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that Far11, a member of the Far3-7-8-9-10-11 complex involved in pheromone-induced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and deletions of components of the Far3-7-8-9-10-11 complex and PP2A rescue growth defects in lst8Δ and tor2-21 mutants. In addition, loss of the regulatory B' subunit of PP2A Rts1 or Far11 restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11 orthologs FAM40A/B exist in a complex with PP2A known as STRIPAK, suggesting a conserved functional association of PP2A and Far11. Antagonism of TORC2 signaling by PP2A-Far11 represents a novel regulatory mechanism for controlling spatial cell growth of yeast.     

A Structural Systems Biology Approach for Quantifying the Systemic Consequences of Missense Mutations in Proteins

Gauging the systemic effects of non-synonymous single nucleotide polymorphisms (nsSNPs) is an important topic in the pursuit of personalized medicine. However, it is a non-trivial task to understand how a change at the protein structure level eventually affects a cell''s behavior. This is because complex information at both the protein and pathway level has to be integrated. Given that the idea of integrating both protein and pathway dynamics to estimate the systemic impact of missense mutations in proteins remains predominantly unexplored, we investigate the practicality of such an approach by formulating mathematical models and comparing them with experimental data to study missense mutations. We present two case studies: (1) interpreting systemic perturbation for mutations within the cell cycle control mechanisms (G2 to mitosis transition) for yeast; (2) phenotypic classification of neuron-related human diseases associated with mutations within the mitogen-activated protein kinase (MAPK) pathway. We show that the application of simplified mathematical models is feasible for understanding the effects of small sequence changes on cellular behavior. Furthermore, we show that the systemic impact of missense mutations can be effectively quantified as a combination of protein stability change and pathway perturbation.     

Phylogeography of Sula: the role of physical barriers to gene flow in the diversification of tropical seabirds

We examined mitochondrial cytochrome b sequence variation in masked Sula dactylatra , red-footed S. sula , and brown S. leucogaster boobies sampled from islands in the central and eastern Pacific Ocean and in the Caribbean Sea. Each species showed a different phylogeographic pattern. Whereas haplotypes in masked and red-footed boobies were shared across the central and eastern Pacific (i.e., across the Eastern Pacific Basin), brown booby haplotypes were not shared across the Eastern Pacific Basin. Although most masked booby haplotypes from the Pacific were distinct from those in the Caribbean, one haplotype was shared across the Isthmus of Panama. Red-footed and brown boobies, however, did not share haplotypes across the Isthmus of Panama. We estimate that divergence of these regional populations occurred within the last 560,000 years. Thus, the Isthmus of Panama and the Eastern Pacific Basin (albeit to a lesser degree) appear to have played a role in the diversification of these species.     

Stigmatic Self-Incompatibility and Mating Patterns in Trillium grandiflorum and Trillium erectum(Melanthiaceae)

Post-pollination processes governing mating patterns in Trillium,a well-known genus of insect-pollinated woodland herbs, arepoorly understood. Mechanisms influencing outcrossing were investigatedin T. grandiflorum and T. erectum, two widespread species nativeto eastern North America. In southern Ontario, Canada, the twospecies are often sympatric; they flower in early May, and arepollinated by different assemblages of insects. Controlled cross-and self-pollinations and structural observations of pollengermination and pollen tube growth were conducted to determinewhether the two species possess a self-incompatibility (SI)system and, if so, the specific site(s) of self-rejection. Controlledpollinations indicated that both species set significantly moreseeds from cross-pollination than self-pollination, implicatingthe action of SI. This was confirmed by structural studies whichdemonstrated that self-recognition and rejection reactions occurredon dry-type stigmatic papillae. Observations of pollen hydrationrevealed that self-rejection was rapid, being initiated within10 min of pollination and prior to pollen tube emergence. Finalself-rejection resulted in failure of pollen tube growth atthe base of stigmatic papillae. SI was expressed more weaklyin T. erectum and thereby resulted in considerable self-seedset in some individuals . Estimates of outcrossing rates usingallozyme markers indicated that T. erectum displayed a mixed-matingsystem whereas T. grandiflorum was more highly outcrossed. Structuralstudies of pollen traits indicated that the two species differedwith respect to the size of grains and their aggregation withimplications for pollen dispersal and mating. The ecologicaland evolutionary implications of the variable expression ofSI in Trillium are discussed. Copyright 2001 Annals of BotanyCompany Trillium grandiflorum, Trillium erectum, self-incompatibility, mating     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.     

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress

Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.