A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae

Hung, Chao‐Wei; Martínez-Márquez, Jorge Y.; Javed, Fatima; Duncan, Mara C.

doi:10.1038/s41598-018-30305-z

Cited by 33 publications

(25 citation statements)

References 57 publications

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“…This assay was conducted as described previously (Hung et al, 2018). Briefly, we initiated cultures at 0.05 OD 600 in each well by appropriately diluting mid-log phase cultures maintaining ∼ 160 μl final volume.…”

Section: Methodsmentioning

confidence: 99%

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

Roy

Joglekar

Fontan

et al. 2020

Preprint

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The Spindle Assembly Checkpoint (SAC) maintains genome stability while enabling timely anaphase onset. To maintain genome stability, the SAC must be strong so that it delays cell division even if one chromosome is unattached, but for timely anaphase onset, it must be responsive to silencing mechanisms. How it meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of 'MELT' motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Spc105/KNL1 must contain many strong MELT motifs to prevent chromosome missegregation, but not too many, because this delays SAC silencing and anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for significantly improved chromosome segregation accuracy. We propose that the necessity of balancing SAC strength with responsiveness drives the evolutionary trend of MELT motif number amplification and degeneration of their functionally optimal amino acid sequence.

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Section: Methodsmentioning

confidence: 99%

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

Roy

Joglekar

Fontan

et al. 2020

Preprint

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“…CFW and sertraline sensitivity assays were performed as previously described (51). Briefly, a log phase culture medium was diluted to 0.02 (A 600 ) in yeast/peptone and transferred to a sterile reagent reservoir.…”

Section: Methodsmentioning

confidence: 99%

Adaptor protein complex-1 (AP-1) is recruited by the HEATR5 protein Laa1 and its co-factor Laa2 in yeast

Zysnarski¹,

Lahiri

Javed³

et al. 2019

Journal of Biological Chemistry

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Edited by Jeffrey E. Pessin Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat-containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized ␥-earbinding sites found in other AP-1-binding proteins. This motif in Laa2 is essential for the Laa1-AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the ␥-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.

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“…We prepared this experiment as described previously (Hung et al, 2018). To set a 96-well plate liquid culture, we used logphase cultures of 0.05 OD 600 as determined by a standard spectrophotometer.…”

Section: -Well Plate Liquid Culture Assaymentioning

confidence: 99%

Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation

Roy

Verma

Sim

et al. 2019

Journal of Cell Biology

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Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore–microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore–microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

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A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae

Cited by 33 publications

References 57 publications

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

Adaptor protein complex-1 (AP-1) is recruited by the HEATR5 protein Laa1 and its co-factor Laa2 in yeast

Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation

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