1. Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function
- Author
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Kristopher M. Locken, Daniel K. Olson, Editte Gharakhanian, Fiona Ruiz, Surya P. Manandhar, and Daniele E. Ejzykowicz
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Endosome ,Mutant ,Saccharomyces cerevisiae ,Golgi Apparatus ,Endosomes ,Vacuole ,Mechanistic Target of Rapamycin Complex 1 ,Protein Serine-Threonine Kinases ,Article ,Phosphatidylinositol 3-Kinases ,03 medical and health sciences ,chemistry.chemical_compound ,Genetics ,Late endosome ,biology ,Qa-SNARE Proteins ,General Medicine ,biology.organism_classification ,Cell biology ,030104 developmental biology ,chemistry ,Biochemistry ,Protein Biosynthesis ,Vacuoles ,Phosphorylation ,Hygromycin B ,VPS45 - 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.
- Published
- 2016