Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MDM33 overexpression suppressor screen

ABSTRACT: We performed a genome-wide screen for genetic MDM33 interaction partners. Using the opposite approach of the well known synthetic dosage lethality (screening for deletion strains in which a non-toxic overexpression is associated with a synthetic growth arrest) we screened the whole genome for genes that are essential for the growth arrest associated with MDM33 overexpression. In a pooled collection of viable haploid gene deletion mutants, of which each is tagged with a unique identifying molecular â??â??bar-codeâ??â?? sequence, the overexpression of MDM33 was induced using a galactose responsive promotor. While overexpression resulted in a growth arrest in almost all strains a small fraction of deletion mutants was able to grow under inductive conditions. We identified these strains using a microarray DNA hybridization technique that quantifies the abundance of the molecular barcodes. We propose that the affected genes are possible functional genetic interaction partners of MDM33. Replicate set 1: knockout strains transformed with empty vector and MDM33 overexpression vector; each transformation plated on glucose and galactose media; uptags and downtags were labelled and detected independently; Replicate set 2: same as replicate set 1, plus dye-swap;

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Alfons Weig

PROVIDER: E-GEOD-49580 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast.

Klecker Till T Wemmer Megan M Haag Mathias M Weig Alfons A Böckler Stefan S Langer Thomas T Nunnari Jodi J Westermann Benedikt B

Scientific reports 20151216

Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed geneti ...[more]

PMID: 26669658

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Project description:The combination of genetic analysis and genome-wide expression profiles make Saccharomyces cerevisiae the cradle of Systems Biology. The way S. cerevisiae uses carbon sources has long been a landmark for studies concerning cellular responses to environmental conditions. S. cerevisiae conserves the entire machinery for utilization of fatty acids, yet with a different compartmentalization compared to higher eukaryotes. We propose S. cerevisiae as a model to understand how perixosomal compartmentalization of oleate metabolism co-evolved with the transcriptional regulatory networks of FA metabolism. With this aim, we performed growth competition experiments in oleate on the pooled collection of all viable yeast null mutants. We integrated results of competitive fitness with transcriptional profiles in oleate, identifying two genes previously not directly associated to FA metabolism: OAR1 and FET3. For the first time we show the importance of iron metabolism not only in maintaining functional mitochondria, but also in influencing peroxisome wellness. An aliquot of the Yeast Homozygous Diploid Deletion Pool (95401.H1Pool, Invitrogen), containing equal amounts of each barcoded viable knock-out mutant from the Saccharomyces Genome Deletion project, was grown in the presence of G418 in YPD medium for 24h, then aliquots of 5x10^7 cells were harvested and stored at -80M-BM-0C. From the same pooled culture, equal amounts of cells were washed and transferred to YP medium added with oleic acid 0.1% and 5%. We collected samples of cells 24h and 72h after carbon source metabolic shift, and genomic DNA was extracted. Each experiment was performed in biological duplicate. The upTAG and downTAG regions were amplified separately in PCR reactions, one for each experimental condition, using specific primers. Amplified upTAG and downTAG sequences were combined in equal amounts and used to probe high-density oligonucleotide arrays (Affymetrix GeneChip_DNA_Tag3). Data analysis was performed as previously described (Pierce et al., 2007). In order to adapt this data analysis protocol to our experimental design, and to the use of GeneChip_DNA_Tag3 arrays, some changes to the procedure have been improved.

Dataset Information

MDM33 overexpression suppressor screen

Publications

Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast.

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