Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

Rothman, Joel H.; Yamashiro, Carl T.; Raymond, Christopher K.; Kane, Patricia M.; Stevens, Tom H.

doi:10.1083/jcb.109.1.93

Cited by 70 publications

(57 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At the same time, the Gef1-containing compartment was slightly acidic with respect to the cytosol and the vacuolar lumen substantially more acidic (Fig. 5B,C,D,E), as expected for these acidifying compartments from the literature (Klionsky et al, 1992;Manolson et al, 1994;Martinez-Munoz and Kane, 2008;Plant et al, 1999;Rothman et al, 1989). Challenge with the iron chelator bathophenanthroline sulfonate (BPS) affected pH homeostasis of all three compartments monitored: in the presence of Gef1, the cytosol became slightly more neutral, the Gef1-containing compartment substantially more neutral and the vacuole more acidic (Fig.…”

Section: Resultssupporting

confidence: 83%

The yeast CLC protein counteracts vesicular acidification during iron starvation

Braun

Morgan

Dick

et al. 2010

Journal of Cell Science

View full text Add to dashboard Cite

SummaryIon gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione -synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.

show abstract

Section: Resultssupporting

confidence: 83%

The yeast CLC protein counteracts vesicular acidification during iron starvation

Braun

Morgan

Dick

et al. 2010

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…The vps-c mutants contain highly fragmented vacuoles and, similar to v-ATPase mutants, have defects in pH homeostasis, acidification of the vacuole, and assembly of the v-ATPase and display alkaline pH sensitivity (Rothman et al 1989b;Sambade et al 2005;Schauer et al 2009;Garipler and Dunn 2013). We first verified that pep3 and pep5 mutants are sensitive to alkaline pH (pH 7.6) ( Figure 5A), consistent with a role for the Vps-C complex in vacuolar acidification and pH homeostasis.…”

Section: Acidic Ph or V-atpase Overexpression Suppresses Rapamycin Sementioning

confidence: 53%

“…VPS-C disruption results in tiny and highly fragmented vacuoles, which are defective in proteolysis and autophagy, thereby significantly reducing both vacuolar amino acid levels and the capacity for vacuolar amino acid storage (Kitamoto et al 1988b;Raymond et al 1992;Klionsky 2005;Liang et al 2008). Third, the acidic pH of the vacuole that is maintained by the v-ATPase is a prerequisite for vacuolar amino acid import and export (Russnak et al 2001), and evidence suggests that vps-c mutants also have defects in vacuole acidification, assembly of the vacuolar v-ATPase, and pH homeostasis (Rothman et al 1989b;Sambade et al 2005;Schauer et al 2009;Garipler and Dunn 2013). Therefore, the multiple mechanisms by which the Vps-C complex maintains cellular amino acid homeostasis likely contribute significantly to regulation of TORC1 signaling.…”

mentioning

confidence: 99%

Endolysosomal Membrane Trafficking Complexes Drive Nutrient-Dependent TORC1 Signaling to Control Cell Growth in Saccharomyces cerevisiae

et al. 2014

View full text Add to dashboard Cite

The rapamycin-sensitive and endomembrane-associated TORC1 pathway controls cell growth in response to nutrients in eukaryotes. Mutations in class C Vps (Vps-C) complexes are synthetically lethal with tor1 mutations and confer rapamycin hypersensitivity in Saccharomyces cerevisiae, suggesting a role for these complexes in TORC1 signaling. Vps-C complexes are required for vesicular trafficking and fusion and comprise four distinct complexes: HOPS and CORVET and their minor intermediaries (i)-CORVET and i-HOPS. We show that at least one Vps-C complex is required to promote TORC1 activity, with the HOPS complex having the greatest input. The vps-c mutants fail to recover from rapamycin-induced growth arrest and show low levels of TORC1 activity. TORC1 promotes cell growth via Sch9, a p70 S6 kinase ortholog. Constitutively active SCH9 or hyperactive TOR1 alleles restored rapamycin recovery and TORC1 activity of vps-c mutants, supporting a role for the Vps-C complexes upstream of TORC1. The EGO GTPase complex Exit from G 0 Complex (EGOC) and its homologous Rag-GTPase complex convey amino acid signals to TORC1 in yeast and mammals, respectively. Expression of the activated EGOC GTPase subunits Gtr1 GTP and Gtr2 GDP partially suppressed vps-c mutant rapamycin recovery defects, and this suppression was enhanced by increased amino acid concentrations. Moreover, vps-c mutations disrupted EGOC-TORC1 interactions. TORC1 defects were more severe for vps-c mutants than those observed in EGOC mutants. Taken together, our results support a model in which distinct endolysosomal trafficking Vps-C complexes promote rapamycin-sensitive TORC1 activity via multiple inputs, one of which involves maintenance of amino acid homeostasis that is sensed and transmitted to TORC1 via interactions with EGOC.T HE Target of rapamycin (TOR) kinases are conserved across eukaryotes and orchestrate myriad cellular processes to control growth in response to nutrients and environmental signals. The Tor kinases form two evolutionarily conserved multi-protein complexes known as TORC1 (Tor complex) and TORC2. TORC1 is sensitive to the immunosuppressive and antiproliferative drug rapamycin, and in Saccharomyces cerevisiae is populated by Tor1 (or, to a lesser extent, Tor2), Kog1, Lst8, and Tco89. TORC1 controls cell growth when nutrients such as amino acids are abundant and serves to maintain robust nutrient transport, ribosome biogenesis, and protein synthesis and concomitantly inhibits autophagy (Heitman et al. 1991;Cardenas et al. 1999;Powers and Walter 1999;Loewith et al. 2002;Reinke et al. 2004;Wullschleger et al. 2006;Loewith and Hall 2011). TORC2 is rapamycin-insensitive and composed of Tor2, Lst8, Bit61/Bit2, Avo1, Avo2, and Avo3; TORC2 controls spatial growth via regulation of actin cytoskeleton polarization (Schmidt et al. 1996;Loewith et al. 2002). Amino acid levels are signaled to yeast TORC1, at least in part, via the leucyl-tRNA synthetase, which binds and influences the guanine nucleotide state of components of the Rag GTPase EGOC (...

show abstract

“…The same is true for ammonium and other weak bases (1,32,55,66). We now present evidence that the charged species CH 3 NH 3 ϩ is accumulated in acidic vacuoles of the yeast S. cerevisiae and the filamentous fungus Neurospora crassa, even when [ 14 C]methylammonium is provided at low external concentrations.…”

mentioning

confidence: 53%

Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH₃ across the Cytoplasmic Membrane

Soupène

Ramirez

Kustu

2001

Molecular and Cellular Biology

View full text Add to dashboard Cite

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH 4 ؉ ؉ NH 3 ). Transport and accumulation of the ammonium analog [14 C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH 4 ؉ . In bacteria, however, ATP-dependent conversion of methylammonium to ␥-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A ⌬vph1 mutant of S. cerevisiae and a ⌬vma1 mutant, which lack vacuolar H ؉ -ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH 3 NH 3 ؉ in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH 3 NH 2 . By inference, their natural substrate is NH 3 , a gas. We postulate that MEP proteins facilitate diffusion of NH 3 across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO 2 .Methylammonium and ammonium permeases MEP1, MEP2, and MEP3 of Saccharomyces cerevisiae (35) and the ammonium and methylammonium transport B (AmtB) protein of enteric bacteria (64) are members of a unique family of cytoplasmic membrane transporters that are specific for ammonium (48). (We use ammonium to designate both the charged and uncharged species.) The MEP/Amt family (nomenclature, TC 2.49) occurs ubiquitously in bacteria, archaea, and eukarya (19,36). Beginning with the pioneering studies of Hackette et al. (16), the activity of MEP/Amt proteins has been assessed by studying transport and accumulation of the ammonium analog methylammonium, which can be 14 C labeled. Based on studies with methylammonium it has been proposed that members of the MEP/Amt family transport the charged species NH 4 ϩ across the cytoplasmic membrane and concentrate it in an energy-dependent manner (19, 65).We showed previously that enteric bacteria convert methylammonium to ␥-N-methylglutamine in the ATP-dependent reaction catalyzed by glutamine synthetase and hence that methylammonium cannot be used to assess energy-dependent concentrative uptake (62). The same metabolic conversion occurs in other proteobacteria, including methylotrophic pseudomonads (2, 1...

show abstract

Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

Cited by 70 publications

References 51 publications

The yeast CLC protein counteracts vesicular acidification during iron starvation

The yeast CLC protein counteracts vesicular acidification during iron starvation

Endolysosomal Membrane Trafficking Complexes Drive Nutrient-Dependent TORC1 Signaling to Control Cell Growth in Saccharomyces cerevisiae

Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH₃ across the Cytoplasmic Membrane

Contact Info

Product

Resources

About

Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins.

Cited by 70 publications

References 51 publications

The yeast CLC protein counteracts vesicular acidification during iron starvation

The yeast CLC protein counteracts vesicular acidification during iron starvation

Endolysosomal Membrane Trafficking Complexes Drive Nutrient-Dependent TORC1 Signaling to Control Cell Growth in Saccharomyces cerevisiae

Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH3 across the Cytoplasmic Membrane

Contact Info

Product

Resources

About

Evidence that Fungal MEP Proteins Mediate Diffusion of the Uncharged Species NH₃ across the Cytoplasmic Membrane