The Pmr1 Golgi Ca²⁺/Mn²⁺ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mM MnCl₂ and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn²⁺ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca²⁺ and Mn²⁺ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn²⁺ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca²⁺ transporting) strain is rapamycin resistant. Mn²⁺ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca²⁺ pump SERCA1, Ca²⁺/H⁺ antiporter Vcx1, or a Mn²⁺ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca²⁺ or Mn²⁺ results in rapamycin resistance. Overexpression of Ccc1, a Fe²⁺ and Mn²⁺ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1. We conclude that Mn²⁺ in the Golgi inhibits TORC1 signaling.

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PMR1 is a member of the P-type ATPase family of ion transporters (RUDOLPH et al. 1989; ANTEBI and FINK 1992). P-type ATPases bind ATP and are phosphorylated at a step in their transport cycles (VANOEVELEN et al. 2005). Pmr1 transports Ca²⁺ or Mn²⁺ ions with high affinity from the cytoplasmic space into compartments of the secretory pathway (RUDOLPH et al. 1989). Pmr1 helps maintain cytoplasmic Ca²⁺ concentration at low values that inhibit signaling to Ca²⁺ effectors until a stimulus, often a cellular stress, induces a transient rise in cytoplasmic Ca²⁺ (CYERT 2003; MULET et al. 2006). A pmr1 mutant increases cytosolic Ca²⁺ and increases Ca²⁺ entry and accumulation in the vacuole. Pmr1 also provides compartments of the secretory pathway lumenally with Ca²⁺ and Mn²⁺. There are possible roles for both ions within the Golgi, including roles in protein modification, in regulation of sorting and vesicular traffic, and in removal of toxic levels of ions. Mannosyltransferases present in the Golgi require Mn²⁺ as a metal cofactor (LISMAN 2004; LOBSANOV et al. 2004). Crystal structures of several glycosyltransferases show Mn²⁺ binds to a conserved DXD motif in the catalytic site (GASTINEL et al. 2001; PERSSON et al. 2001; LOBSANOV et al. 2004). Strains lacking PMR1 have defects in N- and O-linked glycosylations (RUDOLPH et al. 1989; OLIVERO et al. 2003). The secretory pathway is also used to remove metal ions, such as Mn²⁺, that exceed physiologic levels. PMR1 mutants are notably sensitive to high concentrations of extracellular Mn²⁺ (LAPINSKAS et al. 1995).

Recently we reported that Saccharomyces cerevisiae strains with a pmr1 deletion have increased resistance to rapamycin (DEVASAHAYAM et al. 2006). Rapamycin is an immunosuppressive that in complex with Fpr1 (FKBP12) inhibits a subset of functions of the target of rapamycin (TOR) proteins (CRESPO and HALL 2002). There are two TOR genes (TOR1 and TOR2) in S. cerevisiae (reviewed in CRESPO and HALL 2002; DE VIRGILIO and LOEWITH 2006) that encode large (280 kDa) proteins highly conserved throughout evolution that have atypical serine/threonine protein kinase activity, yet are related to phosphatidylinositol 3-kinase protein kinases. TOR2, but not TOR1, is essential in S. cerevisiae. TOR is found in two conserved complexes (TORC1 and TORC2) with distinct composition and function (CRESPO and HALL 2002; DE VIRGILIO and LOEWITH 2006); TORC1, but not TORC2, is inhibited by rapamycin. Deletion of TOR1 consistently causes rapamycin hypersensitivity.

TOR is a central regulator of cell growth and promotes an increase in cell size but not cell number (EDGAR 2006). How TOR regulates cell growth and how nutrient signals regulate TOR are intensely studied problems (WULLSCHLEGER et al. 2006). Given the many cellular processes affected, regulation by TOR of one biological process may be paramount and effects on other cellular processes more indirect. Vesicular trafficking may be a candidate process as it is required for both isotropic and spatially restricted growth and is dependent upon actin dynamics (NEUFELD 2007). Treatment with rapamycin mimics starvation for nitrogen in S. cerevisiae despite availability of nutrients, resulting in endocytosis and degradation of specific permeases and their substitution by the general amino acid permease, Gap1 (CRESPO and HALL 2002). In Drosophila melanogaster, genetic studies of an eye phenotype that results from tissue-specific overexpression of TOR suggest TOR signaling suppresses endocytosis and degradation of specific proteins (HENNIG et al. 2006). Overexpression of Rheb in D. melanogaster, placed in genetic models upstream of TOR, also increased cell-surface expression of Slimfast, a cationic amino acid transporter (HENNIG et al. 2006). In human FL5.12 cells, survival and growth requires cell-surface expression of nutrient transporters whose stability at the membrane requires interleukin-3 (IL3) signaling (EDINGER and THOMPSON 2004). Expression of an allele of mTOR having constitutive activity, but not wild-type mTOR, conferred IL3 independence to FL5.12 by an unidentified mechanism that stabilized nutrient transporters in the absence of IL3 (EDINGER and THOMPSON 2004). These observations strongly indicate that regulation of nutrient transporters by TOR is conserved from yeast to man.

TOR localization is controversial (MARTIN et al. 2006), which may indicate the localization is dynamic and regulated. Studies in yeast and mammalian systems agree that TOR is peripherally associated with intracellular membranes. The localization of mTOR was reported as ER and Golgi (DRENAN et al. 2004) and recently further supported by identification of putative targeting sequences (LIU and ZHENG 2007). In S. cerevisiae, TOR1 has been localized by biochemical fractionation and immunogold electron microscopy (MARTIN et al. 2006). TOR1 was found in fractions with density of plasma membranes and in a lighter, unidentified pool. TOR2 has been reported to be associated with vacuolar membranes (CARDENAS and HEITMAN 1995).

In this article we describe interactions of PMR1 and TOR-dependent signaling pathways. We parse the roles of Ca²⁺ and Mn²⁺ transport by Pmr1 in rapamycin sensitivity. We show that multiple transporters that affect Ca²⁺ and Mn²⁺ do not affect the rapamycin response, suggesting that the effect is specific to Pmr1. We use separation-of-function mutants to show that Mn²⁺ transport by Pmr1 suffices to restore rapamycin sensitivity. We used transporters that are specific to individual compartments to demonstrate that Mn²⁺ transport by Pmr1 into the Golgi is required for rapamycin sensitivity in yeast.

Strains:

The yeast strains used in this study are listed in Table 1, except for strains in Figures 1C and 2A, which are deletion mutants from the haploid collection (replaced by KanMX4) in BY4741 background. The sod1 mutants are in EG103 background (LAPINSKAS et al. 1995). YGD25 (pmr1 tor1) was made by crossing MH349-3d (tor1 in JK9-3da) (HELLIWELL et al. 1994) with LJ25-2C (pmr1 in TB50), sporulated for 2–3 days on KAc plates, and tetrads were dissected. Double mutants were selected as being G418 resistant, grown on leu– plates, and were mating-type .

View this table: In this window In a new window   TABLE 1 Yeast strains used in this study

 

View larger version (51K): In this window In a new window Download PPT slide   FIGURE 1.— Mn2+ suppresses rapamycin resistance of pmr1. (A) pmr1 tor1 grows in the presence of extracellular Mn2+; i.e., pmr1 is synthetically rescued by tor1. tor1 (MH349-3d) is not rapamycin resistant (20 ng/ml) in the presence of extracellular Mn2+ (2 mM) unlike WT (TB50a) and pmr1 tor1 (YGD25). pmr1 tor1 is rapamycin sensitive as is tor1 and WT. Cells were grown for 3–5 days at 30°. (B) Growth of pmr1 strains (LJ25-1A and YGD3) is compared to their WTs (TB50a and BY4741, respectively). TB-WT (TB50a) has a genetic background more sensitive to rapamycin than BY-WT (BY4741) (EUROSCARF). Extracellular Mn2+ suppresses rapamycin (20 ng/ml) resistance of pmr1 (LJ25-1A and YGD3) at 10 µM. Other divalent cations (chloride salts) do not suppress rapamycin resistance of pmr1 at 10 µM. Cells were grown for 3 days at 30°. (C) Mutants of the TOR pathway on rapamycin (20 ng/ml) and 2 mM Mn2+. gln3 is Mn2+ hypersensitive and rapamycin resistant as is pmr1 (YGD3), but rapamycin resistant in the presence of extracellular Mn2+ (2 mM), unlike pmr1. tor1 (BY4741) is sensitive in the presence of rapamycin and Mn2+ (2 mM). Cells were grown for 3 days at 30°. WT: BY4741. Deletion mutants are from the EUROSCARF deletion collection in BY4741 background. (D) sod1 sod2 (EG133) is rapamycin (20 ng/ml) resistant compared to WT (EG103) and Mn2+ (2 mM) enhances this phenotype. Deletion of PMR1 in sod1 sod2 (PJKP-1) also enhances the rapamycin-resistant phenotype.

 

View larger version (64K): In this window In a new window Download PPT slide   FIGURE 2.— Mn2+ transport into the Golgi is required for rapamycin-sensitive signaling in yeast. (A) Of the Ca2+ and Mn2+ transporters in a yeast cell, only deletion of PMR1 confers rapamycin resistance. Deletion mutants were from the EUROSCARF yeast deletion collection in BY4741; cells were grown to midlog phase, serially diluted, and spotted on YPD and YPD containing 100 ng/ml rapamycin. (B) A Pmr1p mutant impaired for Ca2+ transport rescues rapamycin sensitivity. Mn2+ transporting mutant (D53A) of Pmr1 defective for Ca2+ transport suppresses rapamycin resistance. WT and D53A, Q783A, and D778A Pmr1 mutants were expressed in pmr1 (YGD3) from endogenous promoter on a YCp plasmid. Cells were spotted on SC –leu and SC –leu media containing 100 ng/ml rapamycin or 2 mM MnCl2 or both.

 

Plasmids:

Plasmids to express pmr1 point mutations D53A, Q783A, and D778A were constructed by subcloning a fragment [D53A (300 bp XbaI), D778A (2.2 kb BamHI–PstI), and Q783A (2.2 kb BamHI–PstI] from the EBc24-2L1 mutants in pRS415 (BOLTON et al. 2002) (gift of Jeff Boeke) into the same sites of YCp pHAC111-PMR1 (DEVASAHAYAM et al. 2006). All three were sequenced and contained the point mutations. Ccc1 was overexpressed using plasmid pOSC10 that contains CCC1 under control of the MET3 promoter and tagged with FLAG epitope (gift of Jerry Kaplan) (LI et al. 2001). Rabbit SERCA1a cDNA (DURR et al. 1998) (gift of Hans Rudolph) is expressed from the constitutive yeast PMA1 promoter in pRS316 (br434). VCX1 and VCX1-M1 cDNA were expressed from high-copy yeast-expression shuttle vector p2UGpd (PITTMAN et al. 2004), containing the strong constitutive GPD promoter. CAX1 and CAX2 were cloned into yeast shuttle vector piHGpd for expression in yeast.

Media:

Rapamycin (Sigma, St. Louis) was in 90% ethanol/10% Tween-20. 1, 2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) and ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA)-containing media were prepared as described and pH adjusted to 6.0 (with 1 N KOH) and 5.5 (0.5 M MES buffer), respectively (DURR et al. 1998; WEI et al. 2000). Mn²⁺ plates contained MnCl₂; Ca²⁺ plates, CaCl_2. For spottings, cells were 10-fold serially diluted from an initial OD₆₀₀ of 1.0. Yeast transformations were done and standard yeast media prepared as described (AMBERG et al. 2005).

Western blotting:

CCC1 expression was induced in pmr1 (LJ25-1A/a) in the absence of methionine and detected by immunoblot with mouse anti-FLAG M2 antibody (Sigma) at 0.5 µg/ml. Cells were grown in 5 ml SC –leu or SC –leu met medium and protein was extracted by the alkali lysis method (KUSHNIROV 2000). Lysis buffers contain 350 µg/ml p-methyl sulfonyl fluoride, 1x. Complete protease inhibitor (Roche, Indianapolis) and 1 µM phosphatase inhibitor (microcystin-LR).

tor1 suppresses the manganese sensitivity of pmr1:

Pmr1 is a negative regulator of TORC1 signaling (DEVASAHAYAM et al. 2006). Since loss of TOR1 causes rapamycin hypersensitivity (T. F. CHAN et al. 2000), we looked for synthetic interactions of tor1 with pmr1. Throughout this article, we infer TORC1 signaling from how cells respond to the drug. Reduction of TORC1 signaling results in rapamycin resistance and enhanced TORC1 signaling results in rapamycin sensitivity. Strains with a pmr1 mutation do not grow on media containing millimolar concentrations of the divalent cation Mn²⁺ (LAPINSKAS et al. 1995). The Mn²⁺ sensitivity of pmr1 cells was completely suppressed by deleting TOR1 (Figure 1A), suggesting Mn²⁺ sensitivity of Pmr1 was due to hyperactive Tor1. In addition, tor1 pmr1 was sensitive to rapamycin unlike pmr1. Thus, deletion of TOR1 restored the wild-type phenotype in each case. Namely, loss of TOR1 restored the ability of the pmr1 strain to grow on Mn²⁺-containing media such as WT, and loss of TOR1 restored the rapamycin sensitivity of the pmr1 strain to wild-type sensitivity. Interestingly, rapamycin sensitivity of wild type (TB50a) and tor1 pmr1 was partially suppressed in media containing 2 mM Mn²⁺ (Figure 1A).

These data are consistent with a proposed genetic model in which Pmr1 is a negative regulator of TORC1 signaling (DEVASAHAYAM et al. 2006) and suggest that the mechanism may be through regulating manganese homeostasis. Intracellular Mn²⁺ increases dramatically in pmr1 because Mn²⁺ fails to enter the secretory pathway, whereby excess Mn²⁺ can be removed (CULOTTA et al. 2005). Since TOR1 is required for Mn²⁺ toxicity in pmr1, TORC1 appears toxic in this circumstance. Smaller increases in TORC1 activity by Mn²⁺, in the absence of pmr1 or in the presence of pmr1 with tor1, may allow weak growth in the presence of rapamycin.

Extracellular Mn²⁺ increases rapamycin resistance:

TOR proteins are members of the phosphatidylinositol 3-kinase-related protein kinases that include ataxia-telangiectasia mutated (ATM) (WULLSCHLEGER et al. 2006). Both the TOR proteins (ALARCON et al. 1999) and ATM (D. W. CHAN et al. 2000) have been reported to require Mn²⁺ as a cofactor with ATP for maximal activity. Mn²⁺ entry into cells is suppressed by feedback inhibition of expression of the Mn²⁺ transporter Smf1p, but high concentrations of Mn²⁺ still favor entry by alternative, less efficient mechanisms allowing Mn²⁺ to reach toxic levels in pmr1 (CULOTTA et al. 2005).

To distinguish whether low or high extracellular Mn²⁺ was required to increase rapamycin resistance, we studied the dose response for Mn²⁺ in two genetic backgrounds, BY4741 and TB50a (Figure 1B). TB50a is more rapamycin sensitive than BY4741, possibly due to the greater number of auxotrophies for amino acids. The highest concentration of Mn²⁺ tested, 250 µM, increased growth of wild-type strains TB50a and BY4741 on rapamycin but the lower micromolar concentrations of Mn²⁺ did not (data not shown). The toxic effect of Mn²⁺ on growth of the pmr1 strain was detectable at 10 µM. Rapamycin resistance of pmr1 is suppressed by 10 µM Mn²⁺, in both strain backgrounds. To rule out that this effect may be due to osmolarity, we tested the effect of other divalent cations, Ca²⁺, Mg²⁺, Cu²⁺, and Zn²⁺, at the same concentration on pmr1 cells. Only Mn²⁺ suppressed the rapamycin resistance of pmr1 cells (Figure 1B). Mn²⁺ can replace Ca²⁺ in media for growth in some circumstances (LOUKIN and KUNG 1995). Additions of Ca²⁺ to media (1–10 mM) did not increase rapamycin resistance of BY4741 or TB50a (supplemental data at http://www.genetics.org/supplemental/).

The increase in rapamycin resistance by extracellular Mn²⁺ requires GLN3:

Yeast treated with rapamycin act as if starved of nitrogen despite the presence of a good nitrogen source in media. Cells respond to rapamycin by inducing transcription of genes for use of alternative nitrogen sources and by expressing active permeases such as Gap1 to scavenge nitrogen. Gln3, Npr1, and Tip41 positively regulate Gap1 in response to poor nitrogen sources (MAGASANIK and KAISER 2002), and their activities are inhibited by TORC1 signaling. Deletions of these genes confer rapamycin resistance. Nuclear Gln3 transactivates genes including GAP1 that enable adaptation to poor nitrogen sources. A serine/threonine protein kinase, Npr1, positively regulates Gap1, and negatively regulates the more specific amino acid permeases, by control of their sorting and stability (DE CRAENE et al. 2001). Nuclear translocation of Gln3 requires Sit4 phosphatase (BECK and HALL 1999). Tip41 is a Sit4 regulator, and its loss prevents Sit4-dependent dephosphorylation and activation of Npr1 (JACINTO et al. 2001). We compared the effect of Mn²⁺ on the wild-type BY4741 strain of S. cerevisiae and otherwise isogenic strains with deletions in these genes (Figure 1C).

All of these strains grew normally on media containing 2 mM Mn²⁺ with the exception of pmr1 and gln3. Surprisingly, both the pmr1 strain and the gln3 strain were very sensitive to Mn²⁺. On media containing rapamycin, loss of PMR1 induced resistance to a similar degree as loss of NPR1, GLN3, or TIP41. [Deletion of FPR1 (FKBP12) serves as benchmark for rapamycin resistance.] On media additionally containing Mn²⁺, rapamycin resistance was increased for the wild-type BY4741 strain, confirming the observation (Figure 1A) with TB50a. The suppression of rapamycin sensitivity of BY471 by Mn²⁺ (compare YPD + Rap + Mn²⁺ to YPD + Rap) was reduced by gln3 but not by npr1 or tip41. Moreover, the growth defect of gln3 on Mn²⁺ media is partially TORC1 dependent because rapamycin decreases the Mn²⁺ sensitivity of gln3 (compare YPD + Rap + Mn²⁺ to YPD + Mn²⁺). Extracellular Mn²⁺ was unable to suppress rapamycin hypersensitivity of tor1.

A pmr1 mutant is a suppressor of the aerobic growth defect that occurs in a strain lacking Sod1 (LAPINSKAS et al. 1995), a Cu²⁺ containing superoxide dismutase localizing to the cytoplasmic compartment. Suppression is due to an increase of cellular Mn²⁺ to scavenge reactive oxygen species (SANCHEZ et al. 2005). A large screen identified sod1 from the genome-deletion set as having rapamycin resistance (XIE et al. 2005). We tested a sod1 sod2 strain (gift of Valerie Culotta), lacking both cytoplasmic (Sod1) and mitochondrial superoxide dismutase (Sod2), for rapamycin resistance (Figure 1D). Indeed, the sod1 sod2 strain was slightly more resistant to rapamycin in comparison to its wild type (EG103). Either extracellular Mn²⁺ or deletion of PMR1 suppressed the growth defect of sod1 sod2 on rapamycin. This result implicates a reciprocal role of reactive oxygen species (ROS) and Mn²⁺ in TORC1 signaling.

Pmr1-specific Mn²⁺ transport into Golgi is essential for TORC1 signaling:

Pmr1 affects both calcium and manganese homeostasis (RUDOLPH et al. 1989; ANTEBI and FINK 1992). To determine which divalent cation is responsible for the rapamycin responses we compared the pmr1 strain to strains deleted for other cation transporters. Cells from mutants lacking the vacuolar Ca²⁺ transporter gene PMC1 (CUNNINGHAM and FINK 1994), the plasma membrane Mn²⁺ transporter SMF1 (LIU and CULOTTA 1999), the vesicular Mn²⁺ transporter SMF2 (PORTNOY et al. 2000), or the ER-localized P-type ATPase SPF1 (CRONIN et al. 2002) all had the same rapamycin sensitivity as a wild-type-cells strain (Figure 2A). Interestingly, only pmr1 conferred rapamycin resistance. The P-type ATPase Spf1 is involved in Ca²⁺ homeostasis (CRONIN et al. 2002), but did not affect rapamycin sensitivity, suggesting that manganese is the important cation that modulates the response to rapamycin.

Separation-of-function Pmr1 mutants implicate Mn²⁺ in TORC1 signaling:

Point mutations of PMR1 that confer selective transport for Ca²⁺ or Mn²⁺ have been described (WEI et al. 1999, 2000; MANDAL et al. 2000). The mutations were identified by screening for suppression of Ca²⁺ or Mn²⁺ phenotypes in pmr1 pmc1 cnb1 cells. The D53A mutant rescued the Mn²⁺ phenotype and Q783A rescued the Ca²⁺ phenotype, and both Q783A and D53A Pmr1 were localized to Golgi (WEI et al. 2000). D778A is nonfunctional and serves as an additional control. We compared mutants D53A and Q783A to determine if preferential loss of Ca²⁺ or Mn²⁺ transport into Golgi was more important for the rapamycin resistance of pmr1 (Figure 2B). The pmr1D53A cells (defective for Ca²⁺ transport) were rapamycin sensitive, whereas the strains transformed with pmr1Q783A cells (defective for Mn²⁺ transport) and D778A (nonfunctional) mutants were rapamycin resistant. D53A transports Mn²⁺ normally, and it suppressed Mn²⁺ toxicity as did wild-type Pmr1 (Figure 2B). To confirm that pmr1Q783A was functional, we determined that it suppressed BAPTA sensitivity, the Ca²⁺ phenotype (supplemental data at http://www.genetics.org/supplemental/).

Manganese in the Golgi modulates TOR activity:

We used transporters that are specific to individual compartments to demonstrate that Mn²⁺ transport by Pmr1 into the Golgi is required for rapamycin sensitivity in yeast. To rule out the lack of transport of cytosolic Ca²⁺ into the secretory pathway as the cause of rapamycin resistance, we also studied pmr1 cells expressing mammalian SERCA1. Notably, SERCA1 did not complement the rapamycin resistance of pmr1 (Figure 3A). This P2-type Ca²⁺ ATPase restores growth of a pmr1 strain in EGTA- or BAPTA-containing media (DURR et al. 1998; DEGAND et al. 1999). In contrast, SERCA1 was unable to restore growth of pmr1 in high Mn²⁺ (TON and RAO 2004). This suggests rapamycin resistance of pmr1 is not due to entry of Ca²⁺ into the secretory pathway.

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 3.— Ca2+ transport into the secretory pathway is not required for rapamycin sensitivity (A) Normalizing intracellular Ca2+ with SERCA1a does not suppress rapamycin resistance of pmr1. YGD3 was transformed with PMR1 (pKC21, gift of K. Cunningham) or SERCA1a expressed on a YCp vector with yeast PMA1 promoter (br434) or control plasmid (DURR et al. 1998) (gift of H. Rudolph). Cells were serially diluted and spotted; media were as labeled. (B) Vacuolar Mn2+ or Ca2+ transport does not suppress rapamycin phenotype of pmr1. VCX1-M1, Mn2+ transporting mutant of vacuolar H+/Ca2+ antiporter VCX1, suppresses Mn2+ hypersensitivity of pmr1 (YGD3) yet does not restore rapamycin sensitivity. Overexpression of VCX1 in pmr1 also does not restore rapamycin sensitivity; rapamycin, 20 ng/ml; MnCl2, 2 mM. Cells were fivefold serially diluted from a starting OD600 of 2.0. Plates were incubated for 5 days at 30°. WT: BY4741.

 
VCX1 encodes the Ca²⁺/H⁺ exchanger in the vacuolar membrane (PITTMAN et al. 2004). An L208P mutant (VCX1-M1) has enhanced Mn²⁺/H⁺ exchange, and this mutant suppresses the Mn²⁺ toxicity phenotype of pmr1 much better than Vcx1p (PITTMAN et al. 2004). High-dosage expression of neither Vcx1p nor the Mn²⁺ selective mutant L208P suppressed rapamycin resistance due to loss of Pmr1 (Figure 3B). This result suggests that lowering cytosolic Ca²⁺ or Mn²⁺ by transport to the vacuolar space does not restore rapamycin sensitivity of pmr1. The Arabadopsis thaliana CAX1 and CAX2 genes encode Ca²⁺/H⁺ exchangers that localize to the yeast vacuole (SHIGAKI et al. 2003). Cax2 transports Mn²⁺ better than Cax1, and expression of Cax1 and Cax2 decreases the Mn²⁺ toxicity phenotype of pmr1 (SHIGAKI et al. 2003). Neither Cax1 nor Cax2 restored rapamycin sensitivity of pmr1 (supplemental data at http://www.genetics.org/supplemental/), confirming that lowering Ca²⁺ or Mn²⁺ in the cytoplasmic compartment by transport into the vacuole was not sufficient to restore rapamycin sensitivity. Together these data support our conclusion that cytosolic Mn²⁺ does not regulate TOR activity and suggest that the regulation is achieved in a different cellular compartment.

CCC1 restores rapamycin sensitivity:

Pmr1 has been localized to the Golgi (RUDOLPH et al. 1989; ANTEBI and FINK 1992). To determine if the manganese-dependent regulation of TORC1 signaling was dependent on the Golgi, we analyzed the effect of overexpressing CCC1 in pmr1 cells. CCC1 was isolated as a high-dosage suppressor of Mn²⁺ sensitivity of a cnb1 strain (POZOS et al. 1996) and was isolated in an independent screen as a suppressor of the marked Mn²⁺ sensitivity of pmr1 (LAPINSKAS et al. 1996). Furthermore, Ccc1 has been reported to localize to the Golgi (LAPINSKAS et al. 1996). We asked if CCC1 could complement rapamycin resistance of pmr1 (Figure 4). Excess expression of Ccc1p from the inducible MET3 promoter in the pmr1 strain restored wild-type sensitivity to rapamycin, fully complementing pmr1 for this phenotype. We conclude that Mn²⁺ in the Golgi inhibits TORC1 signaling.

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 4.— Interactions of PMR1 with genes involved in Mn2+ homeostasis. (A) Ccc1, a putative Fe2+/Mn2+ transporter (LI et al. 2001), suppresses the rapamycin-resistant phenotype of pmr1 (LJ25-1A) in SC –leu, met medium containing 20 ng/ml rapamycin. Cells were 10-fold serially diluted from a starting OD600 of 5.0. (B) CCC1 expression from the pMET3-CCC1-FLAG plasmid (pOSC10) is induced in SC –leu met medium (in the absence of methionine) but not in SC –leu medium in the LJ25-1A strain background. Mouse anti-FLAG antibody detects Ccc1 tagged with FLAG epitope.

 

In this study we show that TOR1 is required for Mn²⁺ toxicity of pmr1, consistent with our model that Pmr1 is a negative regulator of TORC1 signaling (DEVASAHAYAM et al. 2006). Extracellular Mn²⁺ also improved growth of wild-type and pmr1 tor1 cells. Since some phosphatidylinositol 3-kinase-related protein kinases are Mn²⁺ dependent, this suggested that an increase in cytosolic Mn²⁺ increases Tor1 activity, bypassing rapamycin inhibition. Experiments to parse Ca²⁺ and Mn²⁺ in rapamycin resistance and define the compartments in which Ca²⁺ or Mn²⁺ were important led to the following conclusions:

1. Loss of Mn²⁺ transport appears more important than loss of Ca²⁺ transport for rapamycin resistance in a pmr1 strain.
   
2. Restoration of rapamycin sensitivity requires Mn²⁺ transport into the Golgi or into a vesicular compartment in communication with Golgi.
   
3. Vacuolar Vcx1p-M1 sufficed to suppress Mn²⁺ sensitivity of pmr1 but failed to restore rapamycin sensitivity, disfavoring our initial hypothesis that an increase in cytosolic Mn²⁺ bypasses rapamycin to activate Tor1. The new hypothesis is that an increase in Golgi Mn²⁺ inhibits TORC1 signaling.
   

There were two surprising observations. First, loss of TOR1 suppresses the Mn²⁺ sensitivity of pmr1. Since PMR1 is placed by epistasis upstream of TORC1 signaling (DEVASAHAYAM et al. 2006), the Mn²⁺ sensitivity of pmr1 can be rationalized as Mn²⁺ hyperactivity of Tor1 occurring in this context. Extracellular Mn²⁺ increased rapamycin resistance of pmr1 tor1. Together the data suggest Mn²⁺ regulates both Tor1 and Tor2 in yeast. Whether it is direct or indirect will have to be determined. Second, loss of GLN3 made cells sensitive to Mn²⁺, and sensitivity was reduced by rapamycin showing that the Mn²⁺ sensitivity of gln3 is in part TORC1 dependent. GLN3 target genes include a number of ion transporters in addition to the set of genes for scavenging nitrogen. ENA1, encoding a P-type ATPase of the plasma membrane that transports monovalent cations (Na⁺ and Li⁺), is a Gln3 target and its transcription is induced by rapamycin (CRESPO et al. 2001). PMR1 may also be a Gln3 target gene, since gln3 has a pmr1 phenotype of Mn²⁺ hypersensitivity.

Ccc1p is a putative transporter for Fe²⁺ and Mn²⁺ (LI et al. 2001). Ccc1 has significant similarity to archaebacteria proteins (NP_393546, YP_023568, and NP_110542) including a DMIYGISDGL motif similar to DLIIGLSDGL in Ccc1 that could coordinate a metal ion. Ccc1p is implicated in Fe²⁺ ion homeostasis from the literature but not as a Ca²⁺ transporter. Low cytosolic Fe²⁺ represses CCC1 transcription and negatively regulates CCC1 mRNA by inducing CTH2 (PUIG et al. 2005). High iron in media inhibits growth of a ccc1 strain, and high-dosage expression of CCC1 is protective of iron toxicity (CHEN and KAPLAN 2000). High-dosage expression of Ccc1p-FLAG can increase iron and manganese content in a purified vacuolar fraction, and Ccc1p-FLAG is concentrated at the periphery of the vacuole (LI et al. 2001). Ccc1p has been localized to the Golgi as well (LAPINSKAS et al. 1996), and the latter is important for our interpretation. Both localizations are likely correct, and trafficking of Ccc1p between the Golgi and the vacuole is a distinct possibility. Specific factors may determine which localization predominates. Even if Ccc1 is in the vacuole, it does not preclude it from functioning in the Golgi. Ellis et al. show vacuolar zinc transporters functioning in the ER (ELLIS et al. 2004). Transporters may have activity to transport metals into the lumen of the secretory pathway as they are being trafficked to their final destination.

There are two good ways to imagine how Mn²⁺ in the Golgi regulates rapamycin sensitivity in yeast. The first is that Mn²⁺ regulates permease routing or function. One candidate from what we know from the literature is mannosylation reactions of proteins or lipids (LISMAN 2004). Glycosylations may affect sorting and plasma membrane localization of specific proteins (PROSZYNSKI et al. 2004). Mannosylation of proteins requires Mn²⁺, and this suggests that mannosylation of lipids, as with proteins, could require Mn²⁺. Indeed, sphingolipid mannosylation in yeast requires Csg2-dependent Mn²⁺ transport into the lumen of early secretory organelles, and conversion of inositol phosphoryl-ceramide (IPC) to mannosylinositol phosphoceramide in a csg2 strain is partially suppressed by the addition of millimolar Mn²⁺ to media (LISMAN 2004). IPC is synthesized in the medial Golgi from ceramide precursors made in the ER. Sphingolipids play an important role in sorting proteins, including nutrient permeases, destined for the plasma membrane via formation of detergent-resistant membrane microdomains (PROSZYNSKI et al. 2005). The second possibility is that some TOR may be localized to the Golgi, as suggested for mammalian Tor (LIU and ZHENG 2007), where there is access to Mn²⁺ within the secretory pathway. Further work is necessary to define how Mn²⁺ is involved in sensitivity to rapamycin in yeast.

We thank Jerry Kaplan for discussions and for suggesting our CCC1 experiment, as well as Valerie Culotta, Robbie Loewith, Kendal Hirschi, Jeff Boeke, and Rajini Rao for reagents and/or advice. Michael Hall made helpful suggestions for structuring the manuscript. Hans Rudolph suggested the Mn²⁺ supplement experiment. Support from the Department of Pharmacology is acknowledged. This work was supported by National Institutes of Health grants GM62890 (T.W.S.) and GM40334 (D.J.B.).

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