Multiple TORC1-Associated Proteins Regulate Nitrogen Starvation-Dependent Cellular Differentiation in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth

Hsf1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis

TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections

Multiple Signals Converge on a Differentiation MAPK Pathway

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors

Paclitaxel-polyurethane film for anti-cancer drug delivery: Film characterization and preliminary in vivo study

Polyurethane (PU) films incorporated with an anti-cancer drug paclitaxel (PTX) were prepared using a solvent casting method for potential applications to stent-based drug delivery and the local treatment of malignant tumors around gastrointestinal stents. The films were examined by scanning electron microscopy (SEM), and PTX micro-aggregates were observed when the drug loading was > 2.7 wt%. The in vitro release study revealed that the amount of drug released from the film was virtually independent and cumulative percentage release was inversely proportional to the drug loading. When plotted against the square root of time, the cumulative percentage release was initially linear, but the fraction of the linear region decreased with increasing drug loading, indicating that diffusion-controlled release is not applicable to the PTX molecules in micro-aggregates. When 1.25% PTX-PU films were placed under pre-existing CT-26 tumors in mice, tumor growth was slowed by an average of 65.5% compared to that in the control group.Guo QY, 2009, J CONTROL RELEASE, V137, P224, DOI 10.1016/j.jconrel.2009.04.016Shinke T, 2009, INT J CARDIOL, V135, P93, DOI 10.1016/j.ijcard.2008.06.030Stone GW, 2009, NEW ENGL J MED, V360, P1946Han JK, 2009, MACROMOL RES, V17, P99Lao LL, 2008, J CONTROL RELEASE, V130, P9, DOI 10.1016/j.jconrel.2008.05.008Kim JH, 2008, J VASC INTERV RADIOL, V19, P220, DOI 10.1016/j.jvir.2007.09.023Elkharraz K, 2006, INT J PHARM, V314, P127, DOI 10.1016/j.ijpharm.2005.07.028Zilberman M, 2005, ACTA BIOMATER, V1, P615, DOI 10.1016/j.actbio.2005.06.007Shikanov A, 2005, J CONTROL RELEASE, V105, P52, DOI 10.1016/j.jconrel.2005.02.018Dinnes DLM, 2005, BIOMATERIALS, V26, P3881, DOI 10.1016/j.biomaterials.2004.09.064Lyu SP, 2005, J CONTROL RELEASE, V102, P679, DOI 10.1016/j.jconrel.2004.11.007Lee DK, 2005, GASTROINTEST ENDOSC, V61, P296Khan M, 2005, BIOMATERIALS, V26, P633, DOI 10.1016/j.biomaterials.2004.02.064Stone GW, 2004, NEW ENGL J MED, V350, P221DILOVA V, 2004, B CHIM FARM, V143, P20Han YM, 2003, J VASC INTERV RADIOL, V14, P1291, DOI 10.1097/01.RVI.0000092902.31640.39SINGLA AK, 2002, INT J PHARMACEUT, V235, P197Labow RS, 2001, BIOMATERIALS, V22, P3025Kim DH, 2001, KOREAN J RADIOL, V2, P75Zilberman M, 2001, J BIOMAT SCI-POLYM E, V12, P875Chen KY, 2000, BIOMATERIALS, V21, P161NAXHIMURA B, 2000, HDB PHARM CONTROLLEDLAMBDA NMK, 1997, POLYURETHANES BIOMED

Transcriptomic and biochemical evidence for the role of lysine biosynthesis against linoleic acid hydroperoxide-induced stress in Saccharomyces cerevisiae

Amino acid biosynthesis forms part of an integrated stress response against oxidants in Saccharomyces cerevisiae and higher eukaryotes. Here we show an essential protective role of the l-lysine biosynthesis pathway in response to the oxidative stress condition induced by the lipid oxidant-linoleic acid hydroperoxide (LoaOOH), by means of transcriptomic profiling and phenotypic analysis, and using the deletion mutant dal80Δ and lysine auxotroph lys1Δ. A comprehensive up-regulation of lysine biosynthetic genes (LYS1, LYS2, LYS4, LYS9, LYS12, LYS20 and LYS21) was revealed in dal80Δ following the oxidant challenge. The lysine auxotroph (lys1Δ) exhibited a significant decrease in growth compared with that of BY4743 upon exposure to LoaOOH, albeit with the sufficient provision of lysine in the medium. Furthermore, the growth of wild type BY4743 exposed to LoaOOH was also greatly reduced in lysine-deficient conditions, despite a full complement of lysine biosynthetic genes. Amino acid analysis of LoaOOH-treated yeast showed that the level of cellular lysine remained unchanged throughout oxidant challenge, suggesting that the induced lysine biosynthesis leads to a steady-state metabolism as compared to the untreated yeast cells. Together, these findings demonstrate that lysine availability and its biosynthesis pathway play an important role in protecting the cell from lipid peroxide-induced oxidative stress, which is directly related to understanding environmental stress and industrial yeast management in brewing, wine making and baking

Activity-dependent Reversible Inactivation of the General Amino Acid Permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids