Project description:Comparison between WT Stm1, deltaN terminal and control AP-MS. The pulldown was performed with 3 biological indipendent replicates.
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
Project description:Commercial human heart whole tissue lysates were analyzed with LC-MS/MS with an inclusion list of alternative sequences (Lau et al. bioRxiv 2019).
Project description:Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells preserve a small pool of dormant ribosomes under nutrient-limited conditions. The regulation of dormant ribosomes is poorly characterized. Here, we show that upon inhibition of TORC1 by rapamycin or nitrogen starvation, Stm1 (suppressor of target of Myb protein 1) forms non-translating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon re-feeding, TORC1 directly phosphorylates and inhibits Stm1, thereby reactivating translation. Finally, SERBP1 (SERPINE1 mRNA binding protein), a mammalian ortholog of Stm1, forms dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. Thus, TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner via a ribosome preservation factor.
Project description:A880 has pop2 deletion and a very slow-growth phenotype with glycerol as carbon source. A880 transformed with 2-micron plasmids encoding STM1 can grow robustly on glycerol plates. The Arg237 on Stm1p can be methylated by Hmt1p. A880 transformed with 2-micron plasmids encoding the Stm1p R237K mutant retain the slow-growth phenotype on glycerol plates. We used microarrays to assess the transcription profiles of A880, and A880 transformed with STM1 or STM1 R237K mutant. The goal is to identify genes that are up- or down-regulated in the presence of STM1 R237K mutant but not in the presence of the wild type STM1.
Project description:A880 has pop2 deletion and a very slow-growth phenotype with glycerol as carbon source. A880 transformed with 2-micron plasmids encoding STM1 can grow robustly on glycerol plates. The Arg237 on Stm1p can be methylated by Hmt1p. A880 transformed with 2-micron plasmids encoding the Stm1p R237K mutant retain the slow-growth phenotype on glycerol plates. We used microarrays to assess the transcription profiles of A880, and A880 transformed with STM1 or STM1 R237K mutant. The goal is to identify genes that are up- or down-regulated in the presence of STM1 R237K mutant but not in the presence of the wild type STM1. BY4741 (wild type control), A880 (BY4741 with pop2 deletion), A880STM1 (A880 transformed with STM1) and A880STM1K (A880 transformed with STM1 carrying an Arg237 to lysine substitution) cells were grown in SC medium with glucose as carbon source to early log phase. The cells were shifted to SC medium containing 2% glycerol and incubated for 12 hours before harvesting. Total RNAs were extracted for microarray analysis. Two data sets were generated from separate experiments and cell cultures.
Project description:Targeted SILAC determination of the rate of synthesis of proteins involved in mitochondrial structure and ETC activity in CT26 control and IF1-ablated cell lines