YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information.

The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease.

Elevation of endogenous sphingolipid long-chain base phosphates kills Saccharomyces cerevisiae cells.

Sphingolipid long-chain base phosphates (LCBPs) regulate cell proliferation, movement and differentiation in higher eukaryotes. To study the function of LCBPs in Saccharomyces cerevisiae, we inactivated LCBP breakdown pathways. Elimination of both the Dpll lyase and the Lcb3 phosphatase pathways by gene deletion was lethal, indicating that these enzymes regulate LCBP levels to prevent accumulation. Lethality was prevented by eliminating the major LCB kinase. Lcb4p, which synthesizes LCBPs, but not by eliminating the minor LCB kinase, Lcb5p. These data imply that death results from an accumulation of LCBPs made by the Lcb4p kinase. By regulating Lcb4 kinase activity, we found that cell death correlates with LCBP accumulation and that C18 dihydrosphingosine-l-P (DHS-P) and C20 DHS-P are most likely the killing molecules. LCB levels were found to be most elevated in a strain lacking Lcb4 kinase, Dpll lyase and Lcb3 phosphatase activity. Analysis of mutant strains suggests that the C18 and C20 species of LCBPs are preferentially degraded by the Lcb3 phosphate phosphatase, while the Dpll lyase prefers C16 DHS-P as a substrate. These and other data indicate the existence of an unknown mechanism(s) for regulating LCB levels. Our results demonstrate that LCBPs may be used in some circumstances to regulate yeast cell growth.

Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces.

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44 degreesC heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.

Inhibition of amino acid transport by sphingoid long chain bases in Saccharomyces cerevisiae.

Sphingoid long chain bases have many effects on cells including inhibition or stimulation of growth. The physiological significance of these effects is unknown in most cases. To begin to understand how these compounds inhibit growth, we have studied Saccharomyces cerevisiae cells. Growth of tryptophan (Trp-) auxotrophs was more strongly inhibited by phytosphingosine (PHS) than was growth of Trp+ strains, suggesting that PHS diminishes tryptophan uptake and starves cells for this amino acid. This hypothesis is supported by data showing that growth inhibition is relieved by increasing concentrations of tryptophan in the culture medium and by multiple copies of the TAT2 gene, encoding a high affinity tryptophan transporter. Measurement of tryptophan uptake shows that it is inhibited by PHS. Finally, PHS treatment induces the general control response, indicating starvation for amino acids. Multiple copies of TAT2 do not protect cells against two other cationic lipids, stearylamine, and sphingosine, indicating that the effect of PHS on tryptophan utilization is specific. Other data demonstrate that PHS reduces uptake of leucine, histidine, and proline by specific transporters. Our data suggest that PHS targets proteins in the amino acid transporter family but not other distantly related membrane transporters, including those necessary for uptake of adenine and uracil.