Vitiello SP, Benedict JW, Padilla-Lopez S, Pearce DA
Hum Mol Genet. 2010 Mar 1;19(5):931–42
Juvenile Batten disease is an autosomal recessive pediatric neurodegenerative disorder caused by mutations in the CLN3 gene. The CLN3 protein primarily resides in the lysosomal membrane, but its function is unknown. We demonstrate that CLN3 interacts with SBDS, the protein mutated in Shwachman-Bodian-Diamond syndrome patients. We demonstrate that this protein-protein interaction is conserved between Btn1p and Sdo1p, the respective yeast Saccharomyces cerevisiae orthologs of CLN3 and SBDS. It was previously shown that deletion of BTN1 results in alterations in vacuolar pH and vacuolar (H(+))-ATPase (V-ATPase)-dependent H(+) transport and ATP hydrolysis. Here, we report that an SDO1 deletion strain has decreased vacuolar pH and V-ATPase-dependent H(+) transport and ATP hydrolysis. These alterations result from decreased V-ATPase subunit expression. Overexpression of BTN1 or the presence of ionophore carbonyl cyanide m-chlorophenil hydrazone (CCCP) causes decreased growth in yeast lacking SDO1. In fact, in normal cells, overexpression of BTN1 mirrors the effect of CCCP, with both resulting in increased vacuolar pH due to alterations in the coupling of V-ATPase-dependent H(+) transport and ATP hydrolysis. Thus, we propose that Sdo1p and SBDS work to regulateBtn1p and CLN3, respectively. This report highlights a novel mechanism for controlling vacuole/lysosome homeostasis by the ribosome maturation pathway that may contribute to the cellular abnormalities associated with juvenile Batten disease and Shwachman-Bodian-Diamond syndrome.
Kaufmann I, White E, Azad A, Marguerat S, Bähler J, Proudfoot NJ
Mol Cell Biol. 2010 Jul;30(13):3396–410
Expression of nitrogen metabolism genes is regulated by the quality of the nitrogen supply. Here, we describe a mechanism for the transcriptionalregulation of the general amino acid permease gene per1 in Schizosaccharomyces pombe. We show that when ammonia is used as the nitrogen source, low levels of per1 are transcribed and histones in the coding and surrounding regions of per1 are acetylated. In the presence of proline, per1transcription is upregulated and initiates from a more upstream site, generating 5'-extended mRNAs. Concomitantly, histones at per1 are deacetylated in a Clr6-dependent manner, suggesting a positive role for Clr6 in transcriptional regulation of per1. Upstream initiation and histonedeactylation of per1 are constitutive in cells lacking the serine/threonine kinase oca2, indicating that Oca2 is a repressor of per1. Oca2 interacts with a protein homologous to the Saccharomyces cerevisiae transcriptional activator Cha4 and with Ago1. Loss of Cha4 or Ago1 causes aberrant induction of per1 under noninducing conditions, suggesting that these proteins are also involved in per1 regulation and hence in nitrogen utilization.
Timmermann B, Jarolim S, Russmayer H, Kerick M, Michel S, Krüger A, Bluemlein K, Laun P, Grillari J, Lehrach H, Breitenbach M, Ralser M
Aging (Albany NY). 2010 Aug;2(8):475–86
The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominant Saccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.
Ottosson L-G, Logg K, Ibstedt S, Sunnerhagen P, Käll M, Blomberg A, Warringer J
Eukaryotic Cell. 2010 Oct;9(10):1635–47
Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.
Pang Y, Wang H, Song W-Q, Zhu Y-X
Plant Biol (Stuttg). 2010 Nov;12(6):903–9
The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthaseactivity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors ofATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation.
Strudwick N, Brown M, Parmar VM, Schröder M
Mol Cell Biol. 2010 Dec;30(23):5514–30
Pseudohyphal growth and meiosis are two differentiation responses to nitrogen starvation of diploid Saccharomyces cerevisiae. Nitrogen starvation in the presence of fermentable carbon sources is thought to induce pseudohyphal growth, whereas nitrogen and sugar starvation induces meiosis. In contrast to the genetic background routinely used to study pseudohyphal growth (Σ1278b), nonfermentable carbon sources stimulate pseudohyphalgrowth in the efficiently sporulating strain SK1. Pseudohyphal SK1 cells can exit pseudohyphal growth to complete meiosis. Two stimulators of meiosis, Ime1 and Ime2, are required for pseudohyphal growth of SK1 cells in the presence of nonfermentable carbon sources. Epistasis analysis suggests that Ime1 and Ime2 act in the same order in pseudohyphal growth as in meiosis. The different behaviors of strains SK1 and Σ1278b are in part attributable to differences in cyclic AMP (cAMP) signaling. In contrast to Σ1278b cells, hyperactivation of cAMP signaling using constitutively active Ras2(G19V) inhibited pseudohyphal growth in SK1 cells. Our data identify the SK1 genetic background as an alternative genetic background for the study of pseudohyphal growth and suggest an overlap between signaling pathways controlling pseudohyphal growth and meiosis. Based on these findings, we propose to include exit from pseudohyphal growth and entry into meiosis in the life cycle of S. cerevisiae.
Ghillebert R, Swinnen E, De Snijder P, Smets B, Winderickx J
Biochem J. 2011 Mar 1;434(2):243–51
When starved of P(i), yeast cells activate the PHO signalling pathway, wherein the Pho4 transcription factor mediates expression of genes involved in P(i) acquisition, such as PHO84, encoding the high-affinity H(+)/P(i) symporter. In contrast, transcription of PHO87 and PHO90, encoding the low-affinity H(+)/P(i) transport system, is independent of phosphate status. In the present work, we reveal that, upon P(i) starvation, these low-affinityP(i) transporters are endocytosed and targeted to the vacuole. For Pho87, this process strictly depends on SPL2, another Pho4-dependent gene that encodes a protein known to interact with the N-terminal SPX domain of the transporter. In contrast, the vacuolar targeting of Pho90 upon Pi starvation is independent of both Pho4 and Spl2, although it still requires its SPX domain. Furthermore, both Pho87 and Pho90 are also targeted to the vacuole upon carbon-source starvation or upon treatment with rapamycin, which mimics nitrogen starvation, but although these responses are independent of PHO pathway signalling, they again require the N-terminal SPX domain of the transporters. These observations suggest that other SPX-interacting proteins must be involved. In addition, we show that Pho90 is the most important P(i) transporter under high P(i) conditions in the absence of a high-affinity P(i)-transport system. Taken together, our results illustrate that Pho87 and Pho90 represent non-redundant P(i)transporters, which are tuned by the integration of multiple nutrient signalling mechanisms in order to adjust P(i)-transport capacity to the general nutritional status of the environment.
Caballero A, Ugidos A, Liu B, Oling D, Kvint K, Hao X, Mignat C, Nachin L, Molin M, Nyström T
Mol Cell. 2011 May 6;42(3):390–400
Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.
Zhu X, Liu B, Carlsten JOP, Beve J, Nyström T, Myers LC, Gustafsson CM
Mol Cell Biol. 2011 Jun;31(12):2413–21
The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
Yu S-L, Kang M-S, Kim H-Y, Lee SH, Lee S-K
Mol Cell Toxicol. The Korean Society of Toxicogenomics and Toxicoproteomics; 2011 Oct 5;7(3):195–206
Yeast RAD2, a counterpart of human XPG, is an essential gene for nucleotide excision repair. Rad2p is an endonuclease that incises the 3′ side of a DNA damaged lesion. However, over-expression of Rad2p provokes cell growth arrest, resulting in mitotic catastrophe as evidenced by formation of enlarged cells and micro-nucleation. Interestingly, the effect of Rad2p on cell growth arrest is not caused by its endonuclease activity, and it has been suggested that Rad2p might be involved in cell cycle regulation. Mitotic catastrophe mainly results in cell death but also functions as a survival mechanism. Using RAD2, we show that some yeast cells growth-arrested by Rad2p-induced mitotic catastrophe regain their ability to proliferate mainly because some Rad2p-induced polyploid cells produce haploid cells with normal cell cycles. Rad2p over-expression also causes increased mutagenesis and the cells with recovered proliferative ability exhibit increased genomic instability, which in turn decreases Rad2p expression. From these results it is inferred that Rad2pinduced polyploid cells undergo increased genetic rearrangement resulting in production of haploid cells.