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

Abstract

S. 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 sub-translation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al., 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, 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.

Exomer Is Part of a Hub Where Polarized Secretion and Ionic Stress Connect

“…The pattern of stress sensitivities of mutants defective in clathrin adaptors, whose endosomes are altered ( Kita et al, 2004 ; Hoya et al, 2017 ; Yanguas et al, 2019 ), demonstrated that their PM was hyperpolarized. This agreed with the role of the endosomal system in pH and ion homeostasis, and with results demonstrating a link between defects in the endosomal systems and sensitivity to hygromycin B ( Banuelos et al, 2010 ; Scott and Gruenberg, 2011 ; Ejzykowicz et al, 2017 ). Nevertheless, the growth of mutants lacking exomer and clathrin-associated adaptors in the presence of various salts and sorbitol were different.…”

TORC1 specifically inhibits microautophagy through ESCRT-0

“…In this context, we recently identified two spatially and functionally distinct pools of TORC1 that reside on the limiting membranes of vacuoles and some endosomes that we coined signaling endosomes (Hatakeyama and De Virgilio 2019; Hatakeyama et al 2019). Interestingly, vacuolar TORC1 plays a prominent role in regulating protein synthesis via its proximal effector Sch9 (Ejzykowicz et al 2017; Hatakeyama et al 2019; Jin et al 2014; Urban et al 2007). Endosomal TORC1, in contrast, phosphorylates Atg13 to antagonize initiation of macroautophagy, which occurs at perivacuolar foci called preautophagosomal structures that are in close proximity to endosomal membranes (Hatakeyama et al 2019; Suzuki et al 2013).…”

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

“…Yck3 and Env7 are vacuolar membrane kinases involved in the negative regulation of vacuolar fusion (33,44). Yck3 has several previously established downstream nodes, including Vps41, Vam3, and Mon1p (44)(45)(46)58). Vps41 is a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, involved in membrane tethering (33)(34)(35)(36), and Vam3 is a vacuolar Q-SNARE essential for homotypic fusion (21,39).…”