yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Yeast Strains and Growth Conditions
2.3. Western Blot Analysis
2.4. BioID Experiments
2.5. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
2.6. Phenotypic Growth Tests
2.7. Accession Number
3. Results
3.1. Expression of Rps2-BirA* in S. cerevisiae Strains of the Σ1278b Genetic Background
3.2. Rps2-BirA* Specifically Biotinylates Def1 and Stm1
3.3. Asc1 Inversely Affects Rps2-BirA*-Mediated Biotinylation of the mRNA-Binding Paralogs Sro9 and Slf1
3.4. Asc1 Inversely Affects Rps2-BirA*-Mediated Biotin-Enrichment of Bre5 on the One Hand and Rps3 and Vps30 on the Other Hand
3.5. ASC1 Genetically Interacts with UBP3/BRE5 to Protect Cells Against Heat, Starvation, and Translational Stress
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Plasmid Name | Description | Reference |
---|---|---|
pME2783 | pRS416MET25 with MET25Prom, CYC1Term, URA3, CEN/ARS | [33] |
pME2787 | pRS426MET25 with MET25Prom, CYC1Term, URA3, 2 μm | [33] |
pME4364 | CYC1Term, URA3, CEN/ARS, ASC1 with its native promoter (+500 bp) | [10] |
pME4480 | MET25Prom, CYC1Term, URA3, 2 μm, birA* | [18] |
pME4478 | MET25Prom, CYC1Term, URA3, 2 μm, ASC1-birA* | [18] |
pME4799 | MET25Prom, CYC1Term, URA3, 2 μm, RPS2-birA* | This study |
pUG73 | LEU2 deletion cassette | [34] |
Strain Name | Description | Background | Reference |
---|---|---|---|
RH2817 | MATα, ura3-52, trp1::hisG | Σ1278b | [4] |
RH3510 | MATα, ura3-52, trp1::hisG, asc1-loxP SNR24 | Σ1278b | [3] |
RH3493 | MATα, ura3-52, trp1::hisG, Δarg4::loxP, Δlys1::loxP | Σ1278b | [10] |
RH3520 | MATα, ura3-52, trp1::hisG, asc1-loxP SNR24, ∆arg4::loxP, ∆lys1::loxP | Σ1278b | [10] |
Y06078 | MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; BRE5::kanMX4 | S288c | Euroscarf |
Y06148 | MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; UBP3::kanMX4 | S288c | Euroscarf |
RH3789 | MATα, ura3-52, trp1::hisG, ∆bre5::kanMX4 | Σ1278b | This study |
RH3790 | MATα, ura3-52, trp1::hisG, asc1-loxP SNR24, ∆bre5::kanMX4 | Σ1278b | This study |
RH3791 | MATα, ura3-52, trp1::hisG, ∆ubp3::kanMX4 | Σ1278b | This study |
RH3792 | MATα, ura3-52, trp1::hisG, asc1-loxP SNR24, ∆ubp3::kanMX4 | Σ1278b | This study |
BRE5-GFP | MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0, BRE5-GFP_HIS3MX6 | S288c | Invitrogen, [35] |
RH3793 | MATa, ura3Δ0, leu2Δ0, his3Δ1, MET15, BRE5-GFP_HIS3MX6 | S288c | This study |
RH3794 | MATa, ura3Δ0, leu2Δ0, his3Δ1, MET15, BRE5-GFP_HIS3MX6, ∆asc1::LEU2 | S288c | This study |
No. | Command | Description |
---|---|---|
1 | Generic matrix upload | proteinGroups.txt normalized ratios, etc. |
2.1 | Filter rows based on categorical columns | Remove rows with “+” in reverse column |
2.2 | Remove rows with “+” in potential contaminant column | |
2.3 | Remove rows with “+” in only identified by site column | |
3 | Transform | Inverse ratios (1/x), ratios are reported as follows: ASC1 RPS2-birA*/birA* asc1− RPS2-birA*/birA* asc1− RPS2-birA*/ASC1 RPS2-birA* |
4 | Transform | log2(x) |
5 | Filter rows based on valid values | 9 valid values in total, Reduce matrix (431 proteins remained) |
6.1 | Categorical annotation rows | Group biological replicates 1: ASC1 RPS2-birA*/birA* |
6.2 | 2: asc1− RPS2-birA*/birA* | |
6.3 | 3: asc1− RPS2-birA*/ASC1 RPS2-birA* | |
6.4 | 4: ASC1 RPS2-birA*/birA* and asc1− RPS2-birA*/birA* (for two sample t-test) | |
7.1 7.2 | Two-sample tests | Select the two groups in 4 (see 6.4), p-value threshold: 0.05 p-value threshold: 0.01 |
Filter for proteins enriched from ASC1 RPS2-birA* | ||
8 | Filter rows based on valid values | 3 valid values in group 1 greater than or equal to 0.485 (approximately 40% enrichment), reduce matrix |
9 | Average groups | Calculate mean and standard deviation |
Filter for proteins enriched from asc1− RPS2-birA* but not from ASC1 RPS2-birA* | ||
10 | Filter rows based on valid values | 3 valid values in group 3 outside -0.485 and 0.485 Reduce matrix |
11 | Filter rows based on valid values | 3 valid values in group 2 greater than or equal to 0.485, Reduce matrix |
12 | Average groups | Calculate mean and standard deviation |
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Schmitt, K.; Valerius, O. yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night. Cells 2019, 8, 1384. https://doi.org/10.3390/cells8111384
Schmitt K, Valerius O. yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night. Cells. 2019; 8(11):1384. https://doi.org/10.3390/cells8111384
Chicago/Turabian StyleSchmitt, Kerstin, and Oliver Valerius. 2019. "yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night" Cells 8, no. 11: 1384. https://doi.org/10.3390/cells8111384