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Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae

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Abstract

The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.

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Acknowledgments

We are grateful to Greg Payne for the gift of the deletion strain library, parent vectors, and for continuous support and guidance. We thank Judy Brusslan for technical advice and helpful discussions, and the laboratories of J. Betz, J. Broach, S. Emr, and D. Wolf for generous gifts of plasmids. This work was supported by NIH grant #R15GM1085794-01 to EG. DEM and CRA-S were supported by NIH-RISE and NIH-MARC undergraduate training grants, respectively. DEM was partially supported by the cited NIH-R15 grant. DKO was supported by a Beckman Scholars award.

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  1. Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA

    M. G. Banuelos, D. E. Moreno, D. K. Olson, Q. Nguyen, F. Ricarte, C. R. Aguilera-Sandoval & Editte Gharakhanian

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  1. M. G. Banuelos
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Correspondence to Editte Gharakhanian.

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Communicated by S. Hohmann.

M. G. Banuelos and D. E. Moreno contributed equally to this work.

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Banuelos, M.G., Moreno, D.E., Olson, D.K. et al. Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae . Curr Genet 56, 121–137 (2010). https://doi.org/10.1007/s00294-009-0285-3

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