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

• Published in: Current genetics Vol. 63; no. 3; pp. 531 - 551
• Main Authors: Ejzykowicz, Daniele E., Locken, Kristopher M., Ruiz, Fiona J., Manandhar, Surya P., Olson, Daniel K., Gharakhanian, Editte
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2017; Springer Nature B.V
• Subjects: Biochemistry; Biomedical and Life Sciences; Cell Biology; Defects; Endosomes - drug effects; Endosomes - genetics; Fungi; Golgi apparatus; Golgi Apparatus - drug effects; Golgi Apparatus - genetics; Hygromycin; Hygromycin B; Hygromycin B - pharmacology; Hypersensitivity; Life Sciences; Localization; Lysosomes; Mechanistic Target of Rapamycin Complex 1 - genetics; Microbial Genetics and Genomics; Microbiology; Mutants; Original Article; Phosphatidylinositol 3-Kinases - genetics; Phosphorylation; Plant Sciences; Promotion; Protein Biosynthesis - drug effects; Protein transport; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteomics; Qa-SNARE Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Signal transduction; SNAP receptors; Transduction; Translation; Vacuoles; Vacuoles - drug effects; Vacuoles - genetics; Vps54 protein; TORC1 regulation; Yeast; Trans-Golgi and late endosome interface; Hygromycin hypersensitive mutants; mutants; hhy mutants
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/s00294-016-0660-9

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 h ygromycin B h ypersensitive ( 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 s uper 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.