17 research outputs found
The sensitivity of saccharomyces mutants to palmitoleic acid may provide a means to study the controls of membrane fluidity in eukaryotes
The mechanisms which control the fluidity of eukaryotic membranes are unknown. We have identified S. cerevisiae deletion strains whose growth is impaired by palmitoleic (PO; C16:1) but not oleic (C18:1) acid. PO-sensitivity is suppressed by oleate thus perhaps identifying a signaling pathway that controls the ratio of these fatty acids in membrane phospholipid. Growth of these mutants is also inhibited by a known fluidizer, benzyl alcohol, thus indicating that PO has a fluidizing effect. Removal of Pkc1, known to play a key role in cell wall integrity control, leads to acute PO-sensitivity. Removal of Bck1, Mkk1, Mkk2, Slt2, or Swi6 downstream components of the cell wall integrity pathway, cause modest POsensitivity.
Suppression by 1M sorbitol of the PO-sensitivity of these four mutants implies that PO/oleate ratio influences the cell wall. Acute PO-sensitivity of the pkc1Ξ strain, even in the presence of 1M sorbitol, suggests the cell wall to be more severely compromised by PO addition to this strain.
Alternatively, the failure to control the PO/oleate ratio could have an additional effect on the pkc1 strain, perhaps by disabling a 2nd pathway downstream of Pkc1 thus allowing PO addition to cause excess membrane fluidity. We are attempting to distinguish these two models by a variety of genetic, biochemical, and physical methods. Most notably, the effect of PO on the fluidity of the plasma membrane is being examined by measuring the depolarization of laurdan fluorescence
Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast
Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5β² splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention
The Sensitivity of Yeast Mutants to Oleic Acid Implicates the Peroxisome and Other Processes in Membrane Function
The peroxisome, sole site of Ξ²-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified βΌ130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190Ξ, sit4Ξ, and ilm1Ξ strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids
Rho signaling participates in membrane fluidity homeostasis.
Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation
Losses of Rho1 GEF and GAP activities have opposing effects on PM fluidity.
<p>Using TMA-DPH as a probe, fluorescence anisotropy was performed on wild type, <i>erg6Ξ</i>, <i>tus1Ξ</i>, <i>sac7Ξ</i>, and <i>bck1Ξ ste11Ξ ssk22Ξ</i> (βMK3Ξβ), grown logarithmically in the absence (β) or presence of either 10 Β΅M C18:1 or 10 Β΅M C16:1. Addition of up to 100-fold more C18:1 or C16:1 had no additional effect. Anisotropy values are expressed relative to wild type (in the absence of either soap) and are shown as the mean Β± S.E.M calculated from at least 3 independent experiments. <b>*</b>, <i>p</i>β€0.005 <i>vs.</i> wild type by paired, two-tailed Student's t-test.</p
Structures of C18:1 (oleic acid; C18:1Ξ9) and C16:1 (palmitoleic acid; C16:1Ξ9).
<p>Structures of C18:1 (oleic acid; C18:1Ξ9) and C16:1 (palmitoleic acid; C16:1Ξ9).</p