Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy
Abstract
:1. Introduction
2. Results
2.1. The Sclsm4Δ1 Mutant Shows Regulated Cell Death Markers and Premature Aging
2.2. The Sclsm4Δ1 Mutant Is Defective in Autophagy
2.3. Low Doses of Rapamycin Cannot Protect Cells from Oxidative Stress
2.4. Sclsm4Δ1 Mutant Accumulated Cytoplasmic Autophagy-Related Structures
3. Discussion
4. Materials and Methods
4.1. Yeast Strains, Growth Conditions, and Plasmid Construction
4.2. Viability Assays
4.3. Rapamycin Treatment
4.4. Fluorescence Microscopy
4.5. H2O2 Sensitivity Test
4.6. Glycerol Growth, Caffeine, Acetic Acid, and Rapamycin Sensitivity Test
4.7. Protein Extraction and Western Blot Analysis of Autophagy-Induced Cells
4.8. RNA Extraction, cDNA Syntesis, and Real-Time qPCR for mRNA Expression of ScLSM4
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Genotype | Source |
---|---|---|
MCY4 | MATα, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23 | [58] |
MCY4/Sclsm4Δ1 | MATα, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23 pRS313/Sclsm4Δ1 | This work |
CML39-11A | MATa, ade1-101, his3-Δ1, leu2, ura3, trp1-289 | [8] |
MCY4/ScLSM4 | MATα, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23 pRS313/ScLSM4 | This work |
MCY4/Sclsm4Δ1 pUG36/ATG8 | MATα, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23 pRS313/Sclsm4Δ1, pUG36/ATG8 | This work |
CML39-11A pUG36/ATG8 | MATa, ade1-101, his3-Δ1, leu2, ura3, trp1-289 pUG36/ATG8 | This work |
MCY4/ScLSM4 pUG36/ATG8 | MATα, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23 pRS313/ScLSM4, pUG36/ATG8 | This work |
BMA38 | MATα, ura3-1, leu2-3, -112, ade2-1, can1-100, his3-11, -15, trp1Δ1 | [39] |
BMA38 lsm1Δ | MATα, ura3-1, leu2-3, -112, ade2-1, can1-100, his3-11, -15, trp1Δ1, lsm1Δ::TRP1 | [39] |
Primer Name | Oligonucleotide Sequence |
---|---|
BamH1-ScLSM4/Sclsm4Δ1 Fw | 5’-AAAAAAGGATCCGTACGCAGTCACAATGCGG-3’ |
SacI-ScLSM4 Rv | 5’-GGGGGGAGCTCACCTGTAAACTAAAGGAAAGCTCG-3’ |
SacI-Sclsm4Δ1 Rv | 5’-GGGGGGAGCTCTTATCTTGCAATTTGATAAACTTGATAAAAGTCC-3’ |
Primer Name | Oligonucleotide Sequence |
---|---|
ScLSM4 N-term Fw | 5’-ATTGACCAACGTAGATAACTGGA-3’ |
ScLSM4 N-term Rv | 5’-TACGGCTTTACTGCTCTCAG-3’ |
TDH3 Fw | 5’-CGGTAGATACGCTGGTGAAGTTTC-3’ |
TDH3 Rv | 5′-TGGAAGATGGAGCAGTGATAACAAC-3′ |
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Share and Cite
Caraba, B.; Stirpe, M.; Palermo, V.; Vaccher, U.; Bianchi, M.M.; Falcone, C.; Mazzoni, C. Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy. Int. J. Mol. Sci. 2023, 24, 13708. https://doi.org/10.3390/ijms241813708
Caraba B, Stirpe M, Palermo V, Vaccher U, Bianchi MM, Falcone C, Mazzoni C. Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy. International Journal of Molecular Sciences. 2023; 24(18):13708. https://doi.org/10.3390/ijms241813708
Chicago/Turabian StyleCaraba, Benedetta, Mariarita Stirpe, Vanessa Palermo, Ugo Vaccher, Michele Maria Bianchi, Claudio Falcone, and Cristina Mazzoni. 2023. "Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy" International Journal of Molecular Sciences 24, no. 18: 13708. https://doi.org/10.3390/ijms241813708