Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.
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.
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA.
Daniele E
DE
Ejzykowicz
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA.
Kristopher M
KM
Locken
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA.|Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, Saint Louis, MO, 63108, USA.
Fiona J
FJ
Ruiz
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA.
Surya P
SP
Manandhar
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA.|Inouye Center for Microbial Oceanography, Research and Education, University of Hawaii, Manoa, Honolulu, HI, 96822, USA.
Daniel K
DK
Olson
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA. E.ghara@csulb.edu.
Editte
E
Gharakhanian
D011992:Endosomes / Q000187:drug effects / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology*; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D014176:Protein Biosynthesis / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000235:genetics
D006921:Hygromycin B; D000076222:Mechanistic Target of Rapamycin Complex 1; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D050765:Qa-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C083324:TOR1 protein, S cerevisiae; C530964:SCH9 protein, S cerevisiae
Hygromycin hypersensitive mutants; TORC1 regulation; Trans-Golgi and late endosome interface; Yeast; hhy mutants