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Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function

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Abstract

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121–137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

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Acknowledgements

This project was funded by NIH-AREA research Grant 2R15GM085794-02 to E.G. and NSF-MRI grant DBI0722757 for confocal microscopy. D.E.E. was supported and K.M.L. was partially supported by the above NIH grant; F.J.R. was supported by NIH-RISE grant 5R25-GM071638-07; D.K.O was supported by Beckman Scholars Program. We thank Dr. Greg Payne (UCLA) for the yeast strain library, Dr. Claudio De Virgilio (University of Geneva) for TOR1-3XGFP strain and for plasmid p1462 encoding Sch9-HA, Dr. Christian Ungermann (University of Osnabruck) for PEP12-RFP and VAM3-RFP expressing plasmids.

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  1. Fiona J. Ruiz

    Present address: Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, Saint Louis, MO, 63108, USA

  2. Daniel K. Olson

    Present address: Inouye Center for Microbial Oceanography, Research and Education, University of Hawaii, Manoa, Honolulu, HI, 96822, USA

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

    Daniele E. Ejzykowicz, Kristopher M. Locken, Fiona J. Ruiz, Surya P. Manandhar, Daniel K. Olson & Editte Gharakhanian

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  1. Daniele E. Ejzykowicz
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Correspondence to Editte Gharakhanian.

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Communicated by M. Kupiec.

D. E. Ejzykowicz and K. M. Locken contributed equally to this work.

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Ejzykowicz, D.E., Locken, K.M., Ruiz, F.J. et al. Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function. Curr Genet 63, 531–551 (2017). https://doi.org/10.1007/s00294-016-0660-9

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