Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ)
© Pagán-Mercado et al.; licensee BioMed Central Ltd. 2012
Received: 1 December 2011
Accepted: 18 May 2012
Published: 30 May 2012
Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Δ) in Saccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive this genetically induced stress, this budding yeast up regulates the PKC1 cell wall integrity pathway (CWIP). More recently, our work indicated that TOR, another stress signaling pathway, was down regulated in myo1Δ strains. Since negative signaling by TOR is known to regulate PKC1, our objectives in this study were to understand the cross-talk between the TOR and PKC1 signaling pathways and to determine if they share upstream regulators for mounting the stress response in myo1Δ strains.
Here we proved that TORC1 signaling was down regulated in the myo1Δ strain. While a tor1 Δ mutant strain had increased viability relative to myo1Δ, a combined myo1Δtor1 Δ mutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21 ts lethal phenotype was observed in the myo1Δ strain in contrast to the chs2 Δ strain, a chitin synthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defects very similar to myo1Δ, where the rescue effect was not observed. We observed two pools of Slt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool that is up regulated by heat shock and one that is up regulated by the myo1Δ stress. The cell wall stress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to act as a negative regulator of TORC1 in the myo1Δ mutant. Finally, the repression of TORC1 was inversely correlated with the activation of PKC1 in the myo1Δ strain.
Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in a myo1Δ strain and hence its survival. We found evidence that the PKC1 and TORC1 pathways share a common upstream regulator associated with the cell wall stress sensor WSC1. Surprisingly, essential TORC2 functions were not required in the myo1Δ strain. By understanding how yeast mounts a concerted stress response, one can further design pharmacological cocktails to undermine their ability to adapt and to survive.
The calcium-dependent protein kinase (Pkc1p) and target-of-rapamycin (TOR) signaling pathways are conserved in yeast and other fungi and are important for stress response and fungal survival. In addition to regulating growth and metabolic activity in normal cells, these pathways also regulate the cellular response to transient cell wall stress during the normal yeast life cycle, and during exposure to heat shock, cell wall damage, or other stressors that can compromise cellular integrity [1–3]. Our studies with myosin type II-deficient (myo1Δ) strains of the budding yeast Saccharomyces cerevisiae, which we have characterized previously as stress mutants, showed that the Pkc1p pathway is activated and essential for myo1Δ strain survival [4–6]. It has been our contention that this activation is due to cell wall stress caused by morphological abnormalities in the lateral cell wall and bud neck architecture [7, 8]. In response to cell wall damage, heat shock, and other types of environmental stress, Rho1p activates the PKC1 cell wall integrity pathway (CWIP), which in turn activates Slt2p (Mpk1p), the Serine/Threonine (Ser/Thr) MAPK at the end of this cascade [1–3]. This leads to transcriptional up regulation of cell wall-related genes by the Rlm1p transcription factor [9–12]. In addition to regulating the genetic program for cell wall integrity through the transcription factor Rlm1p [9, 13, 14], Slt2p may also modulate PKC1 activity indirectly by a previously proposed feedback mechanism that phosphorylates and down regulates the Rho1p GDP-GTP Exchange Factor (GEF) Rom2p . Rho1p also functions as the regulatory subunit of Fks1p, a β-1,3-glucan synthase for lateral cell wall fortification .
In prior studies, we have shown that similar to wild-type (wt) cells under stress conditions, the myo1Δ mutant (a genetically induced stress caused by the deletion of myosin II heavy chain that inhibits normal cytokinetic ring assembly) also activates the PKC1 CWIP, but uses a different repertoire of genes [4, 5]. Further characterization of the genes of the myo1Δ mutant at the post-transcriptional level showed that only a subset of cell wall integrity genes was activated. Thus, the myo1Δ mutant may serve as a simplified model for studying the cell wall stress response. Furthermore, we found that translation and ribosome biogenesis were down regulated in the myo1Δ strain . This observation led us to investigate the role of TOR in the myo1Δ strain survival and how it may complement the reduced CWIP response.
Yeast TOR consists of two proteins - Tor1p and Tor2p - which are contained in two protein complexes TORC1 and TORC2 [18, 19]. The TORC1 complex that is sensitive to rapamycin treatment contains proteins Tor1p or Tor2p, Kog1p, Tco89p and Lst8p [18, 20–22]. TORC2 that is resistant to rapamycin treatment contains Tor2p, Avo1p, Avo2p, Avo3p, Bit61p, and Lst8p [18, 20]. Recent subcellular localization studies showed that Tor1p was concentrated near to the vacuolar membrane while Tor2p was predominantly in punctuate structures near to the cytoplasmic surface of the plasma membrane . Their differences in composition, sensitivity to rapamycin, and cellular localization support the idea that they function as two separate complexes [18, 20, 23]. TOR is important for nutrient sensing and is believed to play an important role in life span extension [24–27]. While TOR is conserved structurally and functionally from yeast to human, their roles are not biologically identical and warrant careful characterization of TOR from both species.
The stress sensor proteins Wsc1p, Wsc2p, Wsc3p, Mid2p and Mtl1p are involved in the activation of cell integrity signaling [2, 32–37]. These cell surface sensors span the plasma membrane and are attached to the extracellular cell wall. The Mid2p homologue Mtl1p, that shares 50% sequence identity with Mid2p, appears to have a minor role in PKC1 signaling . These sensors react differently under specific stress conditions . It has been reported that cells lacking WSC1 are hypersensitive to drugs interfering with the cell wall and plasma membrane like Calcofluor white, Congo red, Caspofungin, Chlorpromazine and tea tree oil [1, 38–40]. Additionally, Wsc1p responds to hypo-osmotic and alkaline pH conditions [39, 41]. A mid2 Δ mutant is hypersensitive to pheromone treatment, is hyperresistant to Calcofluor white, tea tree oil and Congo Red, and it senses acidic conditions and vanadate [32, 37, 39, 40, 42–44]. Wsc1p and Mid2p are also involved in the response to heat shock [2, 33, 35, 45]. WSC2 and WSC3 act as suppressors of mutants defective in glycerol synthesis , while Mtl1p is associated with response to oxidative stress and glucose starvation [46, 47]. The Wsc family of proteins and Mid2p have been shown to interact with specific signaling proteins that transmit stress signals from the fungal cell wall sensors to the Pkc1p and TOR signaling pathways. For example, Rom2p, the GEF that regulates Pkc1p, physically interacts with Wsc1p, Wsc2p, and Mid2p to activate the PKC1 CWIP in the response to cell wall stress [36, 48, 49]. To define the nature of these signaling interactions in myo1Δ strains, we demonstrate here that TORC1 and Pkc1p activities were inversely correlated, which suggests cross-talk between the two pathways. Furthermore, we found that TORC1 was down regulated in myo1Δ strains by a mechanism that required expression of Wsc1p but not the other cell wall stress sensors. Surprisingly, Tor2p functions were not essential for survival in myo1Δ cells.
TORC1 activity is down regulated in myo1Δ strains
The phosphorylation state of Npr1p can be deduced from its relative electrophoretic mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blot analysis using an anti-HA antibody. In wt whole cell extracts, Npr1p was detected as a slower-migrating band, which corresponds to the hyperphosphorylated form, Npr1pP (Figure 2B, lane 1). Treatment of wt whole cell extracts with exogenous Calf Intestinal Alkaline Phosphatase (PPase) converted the slower-migrating band to a faster-migrating band which corresponds to the in vitro dephosphorylated form, Npr1p (Figure 2B, lane 2). Treatment of wt cell cultures with rapamycin produced a faster-migrating Npr1p band that co-migrated with the PPase treated band, consistent with the inhibition of TORC1 activity by rapamycin (Figure 2B, lane 3). This experiment established that the activity of TORC1 could be assessed indirectly by observing the relative electrophoretic mobility of Npr1p by SDS-PAGE [50, 53].
The SIT4 gene encodes a protein phosphatase that is responsible for dephosphorylation of Npr1p in vivo during nutrient starvation (Figure 2A) . TORC1 when active, phosphorylates Tap42p, which then binds and keeps Sit4p inactive . Thus, Sit4p activity is negatively regulated by TORC1 . In previous studies, Npr1p was shown to maintain the hyperphosphorylated state in a sit4 Δ mutant treated with rapamycin indicating that its dephosphorylation was directly dependent on Sit4p activity [50, 53]. To establish that dephosphorylation of Npr1p employs the same mechanism in myo1 Δ, we conducted a Western blot analysis of Npr1p in a myo1 Δsit4 Δ strain (Figure 3B). Absence of Sit4p activity in the myo1 Δ strain resulted in the accumulation of the slow-migrating hyperphosphorylated Npr1p. This result supports that the myo1Δ dephosphorylation of Npr1p is via Sit4p.
To analyze this putative cross-talk further, we assayed the relative levels of P-Slt2p in a myo1 Δtor1 Δ strain. In the absence of Tor1p, a myo1 Δtor1 Δ strain maintained significant steady state levels of P-Slt2p at approximately 50% of the myo1Δ- levels (Figure 4B, lane 5), while treatment with rapamycin did not generate a significant change in these levels (Figure 4B, lane 6). A tor1 Δ single mutant activated PKC1 at low levels (Figure 4B, lanes 3 and 4).
To determine if these strains presented a growth defect we tested wt, myo1Δ, tor1 Δ and myo1Δtor1 Δ strains for cell viability using a serial dilution assay (Figure 4C). The myo1Δ strain exhibited a viability range similar to the wt strain. Surprisingly, the tor1 Δ strain showed increased viability through the 102 cells/ml range, consistent with previous studies that showed that Tor1p functions were not essential for cell viability. Despite having P-Slt2p present (Figure 4B, lane 5) the myo1Δtor1 Δ strain presented a reduction in cell viability of approximately four orders of magnitude (Figure 4C, bottom row). Therefore, down regulation of TORC1 appears to be favorable to maintain viability in the myo1Δ strain while a complete absence of Tor1p in this strain is detrimental. These results imply that Tor1p may have a predominant role in the TORC1 functions with less activity attributed to the Tor2p in this complex. However, the residual activity in the TORC1 complex was essential for myo1Δtor1 Δ strain survival because five days treatment with the IC50 of rapamycin (44nM) resulted in a 10-fold further reduction of growth (data not shown).
Positive genetic interaction between MYO1 and TOR2: lethality of a tor2-21 ts allele at 37°C is rescued by myo1 Δ
TORC2 has been shown to have a strong regulatory effect on PKC1 activity in cell wall mutants [29, 30]. To explain the observed synthetic rescue of tor2-21 ts lethality by myo1Δ, we conducted a Western blot analysis of P-Slt2p levels in the wt, myo1Δ (Figure 5C, box1), tor2 Δ ptor2 ts and myo1Δ ptor2 ts strains (Figure 5C, box 2) at the permissive temperature. Wt and tor2 Δ ptor2 ts strains showed similarly low P-Slt2p levels consistent with growth under non-stress conditions (Figure 5C, box 1 and box 2 respectively). The myo1Δ (Figure 5C, box1) and myo1Δ ptor2 ts (Figure 5C, box2) strains both presented a higher level of P-Slt2p relative to wt strains at 26°C, which was also consistent with previous observations that PKC1 is activated in these strains (Figure 4A, lane 3).
To assess if rescue of tor2-21 ts lethality at the restrictive temperature by myo1Δ was accompanied by a change in PKC1 activity levels, P-Slt2p was analyzed in whole cell extracts from cultures taken at 37°C (Figure 5C boxes 3 and 4). The temperature shift to 37°C produced an increase in P-Slt2p levels in all four strains attributable to the heat shock effect that is known to activate the PKC1 pathway [1, 62]. This suggests that there may exist two pools of Slt2p, one that is activated by the myo1Δ mutation and one that is activated (or phosphorylated) by the heat stress. Densitometric quantification and normalization of autoradiographs from duplicate experiments established that P-Slt2p levels in the myo1Δ ptor2 ts strain at 37°C were 5-fold higher than in the tor2 Δ ptor2 ts strain yet were very similar to the myo1Δ single mutant strain at 37°C (Figure 5C, bottom panel). However, when we compared P-Slt2p levels between the tor2 Δ ptor2 ts and myo1Δtor2 Δptor2 ts strain, there was no significant difference between them supporting that the rescue effect was not due to Tor2p-dependent P-Slt2p up regulation (Figure 5C, box 5) or differences in P-Slt2p levels.
Evidence for a cross-talk between Pkc1p, TORC1, and cell wall stress sensor Wsc1p
Relative TORC1 activity levels for the wt and myo1Δ strains were previously shown (Figures 2B and 2C respectively), while the results for myo1Δwsc2 Δ, myo1 Δwsc3 Δ and myo1 Δmid2 Δ strains were also consistent with down regulated TORC1 activity in these strains (data not shown). In contrast, the myo1 Δwsc1 Δ double mutant strain exhibited a result that was consistent with a fully active TORC1 (Figure 6A, lane 1) as judged by the relative decrease in electrophoretic mobility normally exhibited by Npr1pp, and the restored sensitivity of Npr1pp electrophoretic mobility to rapamycin treatment (Figure 6A, lane 2). Also, like the myo1Δ and wsc1 Δ strains (Figure 6A, lanes 3 and 4), preliminary results show that wsc2 Δ, wsc3 Δ, and mid2 Δ single mutant strains (data not shown) exhibited a rapamycin-insensitive Npr1p electrophoretic mobility that was consistent with down regulation of TORC1. These results indicate that absence of these cell wall stress sensors represents a cell stress and supports the idea that they also play a role during normal cell growth. We therefore conclude from these results that Wsc1p may be associated with the regulation of TORC1 in both the wt and myo1Δ strains.
Because TORC1 and PKC1 activities maintain an inverse relationship , we predicted that a re-activation of the TORC1 observed in the myo1 Δwsc1 Δ strain would exert an inhibitory effect on the PKC1 pathway. Consistent with this hypothesis, the myo1 Δwsc1 Δ strain failed to activate the PKC1 pathway as evidenced by undetectable levels of P-Slt2p relative to a myo1Δ single mutant where PKC1 was activated (Figure 6B). Cell viability analysis revealed a reduction in growth of approximately one order of magnitude in the myo1 Δwsc1 Δ double mutant strain relative to the myo1 Δ strain and two orders of magnitude relative to wt and wsc1 Δ strains that grew comparably well (Figure 6C).
Yeast cells must respond rapidly and effectively to alterations in the environment in order to survive stressful conditions. These processes require the involvement of signal transduction pathways such as TOR and PKC1. The PKC1 dependent CWIP is the first line of response to cell wall damage in the yeast Saccharomyces cerevisiae[13, 14]. Transduction of the signal begins with the cell wall stress sensor proteins Wsc1p and Mid2p at the plasma membrane and proceeds through Rom2p and Rho1p to the PKC1 CWIP that ends with activation of the MAP kinase, Slt2p [13, 48] (Figure 1). Downstream, the transcription factor Rlm1p activates nuclear genes involved in cell wall synthesis and remodeling to produce a cell wall stress response that increases the survival potential of the yeast cell [9, 10]. We have shown in prior studies that the PKC1 pathway is continuously activated in myo1Δ strains [4, 6]. This response is further characterized here in the myo1Δ strains. In addition to the up regulation of the CWIP we found that TORC1 was down regulated to enhance cell survival and we provide evidence of cross-talk between the two signaling pathways.
Npr1p is a protein kinase that regulates the amino acid permease Gap1p to transport secondary nitrogen sources into the cell for the restoration of amino acid precursor levels and protein synthesis [51, 64, 65]. When TORC1 is down regulated by nutrient starvation or rapamycin treatment, Npr1p becomes dephosphorylated by the protein phosphatase Sit4p, thereby activating its biochemical function [50, 51, 53] Furthermore, inactivation of TORC1 results in downregulation of ribosome and protein synthesis . When we assayed the relative status of TORC1 activity in a myo1Δ strain, we observed that Npr1p was maintained in the dephosphorylated state and demonstrated that the Npr1p phosphorylation state was directly dependent on TORC1 and Sit4p activities. Therefore, we established that the TORC1 complex is found in a predominantly inactive state in the myo1Δ strain. The implications of such a metabolic state led us to believe that the survival of this strain is directly linked to this observation. However, the complete absence of Tor1p by genetic deletion (tor1 Δ) was detrimental for survival of the myo1Δ strain, supporting that a precise level of TORC1 activity must be maintained for its survival. Furthermore, complete inhibition of TORC1 activity by rapamycin treatment of a myo1Δtor1 Δ strain was lethal for growth, further supporting the idea that minimal levels of TORC1 activity are essential. Conversely, the tor1 Δ single mutant was shown to acquire increased fitness, which was consistent with the proposed role of mTOR and TOR in regulating longevity and replicative life span extension respectively [24–27].
Our findings showed that chs2 Δ and fks1 Δ mutant strains strongly activated the PKC1 CWIP yet maintained normal TORC1 activity levels. We are therefore confronted with variable signaling outputs exiting from the cell wall stress sensors. In particular, disruption of cell wall integrity by these mutants leads to activation of the PKC1 CWIP; however, only the disruption of cytokinesis in a myo1Δ strain leads to both activation of the PKC1 CWIP and down regulation of TORC1 (Figure 8). This finding is consistent with the transcriptional profiles we have determined previously, where myo1Δ only regulated half of the CWIP fingerprint genes, while fks1 Δ and chs2 Δ profiles were more like other cell wall damage profiles [4, 5]. This reinforces the idea that myo1 Δ activates a cell signaling program that is distinct from other cell wall mutants. We propose that in addition to its filament assembly function, the tail domain may serve as a scaffold (or guide) for the assembly of interacting protein complexes at the cytokinetic ring that are important for myosin function . Therefore, disruption of these putative protein assemblies by a genetic deletion of the MYO1 gene may activate the cell wall stress sensors Wsc1p and Mid2p in a different manner than in the chs2 Δ and fks1 Δ mutants. The non-muscle myosin heavy chain (Myo1p) of budding yeast has been shown to have independent functions associated with the head and tail domains of the protein . The tail domain contains a Minimum Localization Domain (MLD) that is sufficient to target the myosin heavy chain to the bud neck independently of the actin-binding site that is encoded within the head domain . Therefore, despite the common activation of the PKC1 CWIP among the myo1 Δ, chs2 Δ and fks1 Δ mutant strains, we hypothesize that the inhibition of TORC1 by Wsc1p is unique to the myo1 Δ mutant and may be triggered by the disruption of specific protein-protein interactions in the putative Myo1p scaffold at the cytokinetic ring.
The final question that arises from these results is, how does the myo1 Δ mutant rescue tor2-21 ts lethality? Strains that carry the temperature-sensitive gene of TOR2 (tor2 ts ) arrest growth at the restrictive temperature (37°C). This lethality is thought to be caused by the lack of TORC2 activity, decreased RHO1 activation , the lack of actin organization and cell lysis probably due to cell wall defects . The lethality has been shown to be rescued in several different ways. One way is by growth on nonfermentative carbon sources (i.e. raffinose) but not by nonfermentable carbon sources (i.e. glycerol or ethanol) . A second way in which tor2 ts lethality can be circumvented is by treatment with agents that cause cell integrity stress (i.e. 0.005% SDS) . A third way in which the tor2 ts lethality can be rescued is by the osmotic stabilizer, sorbitol, again suggesting that the cell wall is somehow compromised. Finally, there are several genes that have been shown to suppress the lethality of tor2 ts lethality. One example is yeast PAS kinase overexpression (a gene involved in glucose partitioning in the cell) which is thought to suppress the tor2 ts lethality by RHO1-dependent activation of PKC1 and actin rearrangement, activation of FKS1 and cell wall synthesis, or both . These observations have lead to the idea that cell growth and survival is a product of signals derived from cell integrity and nutrient availability . In this work we provide evidence for rescue of tor2 ts lethality by the deletion of the MYO1 gene. We propose that myo1 Δ rescues the tor2 ts lethality by invoking both strategies described above, namely, by activating a starvation type response (TOR) and the cell wall integrity pathway (PKC1 CWIP), most likely through the reorganization of the actin cytoskeleton. However, unlike the results of Cardon et al.  where the essential Rho1p GEF was Rom2p, the roles of Rom1p and Rom2p appear to be redundant for the proposed myo1 Δ rescue mechanism (data not shown).
We have shown that cross-talk between the PKC1 and TOR signaling cascades occur under the myo1Δ stress condition. TORC1 activity was found to be inversely correlated with activation of the PKC1 pathway while both Tor1p and Pkc1p act as positive regulators of viability in the myo1 Δ strain. Synthetic rescue of tor2-21 ts lethality by myo1Δ points to the PKC1- dependent reorganization of the actin cytoskeleton as the possible rescue mechanism. The data presented supports that in addition to its known role in signaling to the PKC1 CWIP, Wsc1p may also function as an upstream regulator of TORC1.
Strains and media
Strains used in this study
YJR12,YJR24_1 (wild type, wt)
MAT α trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhR
MAT a leu2-3,112 trp ura3 rmel his4 HMLa
YJR13 (myo1 Δ)
MAT a trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhRmyo1Δ::HIS5+
YFR22 (fks1 Δ)
MAT α trp1-289 ura3-52 leu2-3, 112 his3Δ1 ADE + ARG + cyhRfks1 Δ::kanMX4
YFR23 (chs2 Δ)
MAT α trp1-289 ura3-52 leu2-3, 112 his3Δ1 ADE + ARG + cyhRchs2 Δ::kanMX4
YJR066W (tor1 Δ)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 tor1Δ::kanMX4
YGP1 (myo1 Δtor1 Δ)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 tor1Δ::kanMX4, myo1Δ::HIS5+
YOR008C (wsc1 Δ)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 wsc1Δ::kanMX4
YES1 (myo1 Δwsc1 Δ)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 wsc1Δ::kanMX4, myo1Δ::HIS5+
JK9-3da ade2 tor2::ADE2/pSEY18::TOR2
SH121 (tor2 Δ ptor2ts)
JK9-3da ade2 tor2::ADE2/YCplac111::tor2-21 ts
YGP5 (myo1 Δtor2 Δptor2ts)
JK9-3da ade2 tor2::ADE2/YCplac111::tor2-21 ts myo1 Δ::HIS5+
YJR12/pEJ23 (wt pHA-NPR1)
MAT α trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhR, pHA-NPR1
YJR13pEJ23 (myo1Δ pHA-NPR1)
MAT a trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhR, myo1 Δ::HIS5 + pHA-NPR1
(YFR22pEJ23) (fks1 Δ pHA-NPR1)
MAT α trp1-289 ura3-52 leu2-3, 112 his3Δ1 ADE + ARG + cyhRfks1 Δ::kanMX4, pHA-NPR1
YFR23pEJ23 (chs2 ΔpHA-NPR1)
MAT α trp1-289 ura3-52 leu2-3, 112 his3Δ1 ADE + ARG + cyhRchs2 Δ::kanMX4, pHA-NPR1
YJR066WpEJ23 (tor1 ΔpHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 tor1Δ::kanMX4, pHA-NPR1
YGP1pEJ23 (myo1 Δtor1 Δ pHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 tor1Δ::kanMX4 myo1 Δ::HIS5+, pHA-NPR1
YDL047W pEJ23 (sit4 ΔpHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 sit4Δ::kanMX4, pHA-NPR1
YGP3pEJ23 (myo1Δsit4 Δ pHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 sit4Δ::kanMX4 myo1 Δ::HIS5+, pHA-NPR1
YOR008C pEJ23 (wsc1 ΔpHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 wsc1Δ::kanMX4, pHA-NPR1
YES1 pEJ23 (myo1 Δwsc1 Δ pHA-NPR1)
MAT α his3Δ1 leu2Δ0 lysΔ0 ura3Δ0 wsc1Δ::kanMX4 myo1 Δ::HIS5+, pHA-NPR1
YJR24_1/YCplac111 (wt ptor2 ts )
MAT α trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhR, YCplac111::tor2-21 ts
YJR13/ YCplac111 (myo1 Δ ptor2ts)
MAT a trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhRmyo1 Δ::HIS5+, YCplac111::tor2-21 ts
YFR23/p YCplac111 (chs2 Δptor2ts)
MAT α trp1-289 ura3-52 leu2-3, 112 his3Δ1 ADE + ARG + cyhRchs2 Δ::kanMX4, YCplac111::tor2-21 ts
YJR13YCplac111pRS316MYO1 (myo1 Δptor2tspMYO1)
MAT a trp1 ura3 leu2-3 his3Δ1 met- ADE + ARG + cyhRmyo1 Δ::HIS5+, YCplac111::tor2-21 ts pRS316MYO1
YGP5pRS316MYO1 (myo1 Δtor2 Δptor2tspMYO1)
JK9-3da ade2 tor2::ADE2, myo1 Δ::HIS5+, YCplac111::tor2-21 ts pRS316MYO1
Plasmid and genetic techniques
Plasmid pHA-NPR1 (pEJ23) consists of YEplac181 (LEU2) expressing a functional N-terminally HA-tagged NPR1 under its own promoter , kindly provided by Dr. Estela Jacinto. Plasmid YCplac111::tor2-21 ts (LEU2) containing a temperature sensitive tor2-21 ts allele  was kindly provided by Dr. Michael N. Hall. Escherichia coli strain DH5α was used for the propagation and isolation of plasmids. Yeast transformations were performed by the Lithium acetate procedure. Yeast plasmid DNA was isolated by an adaptation from the QIAGEN QIAprep Spin miniprep kit.
Western blot analysis
Whole yeast cell protein extracts were prepared by harvesting and lysing cell cultures by vortexing with glass beads for 20 s with 3 min intervals on ice (repeated 3 times). Lysis buffer contained 50 mM Tris–HCl pH 7.5, 10% Glycerol, 1% TritonX-100, 0.1%SDS, 150mN NaCl, and 5 mM EDTA, supplemented with 5X Protease Inhibitor Cocktail (50X stock; Roche) and 10 mM PMSF. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C; the supernatant was removed and quantified using the DC Protein Assay method (Bio-Rad, Hercules, CA).
Whole protein extracts were denatured at 95°C for 5 min, separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane at 0.37 Amps for 1 h at 4°C in a Mini Trans Blot Cell (Bio-Rad, Hercules, CA). Npr1p was consistently expressed more abundantly in wt cells than in any of the mutant cells. Therefore, the loading volumes were adjusted accordingly. The reason for these differences in Npr1p levels between strains is not known, but it has been speculated that it could be due to differences in the stability of the protein . For analysis of HA-NPR1, membranes were probed with anti-HA rat monoclonal antibody (3 F10, Roche, 1:1000) in blocking solution containing 0.5% Western Blocking Reagent (Roche) diluted in 1X TBS (Tris Buffered Saline, Sigma Aldrich) at 4°C overnight and washed in 1X TBS/0.1% Tween-20 (TBS/T) (Sigma Aldrich). Membranes were counter-probed with a Horseradish Peroxidase (HRP) conjugated secondary Goat anti-rat IgG antibody (Pierce, 1:5000). For phosphorylated Slt2p (P-Slt2p), membranes were incubated with anti-phospho-p44/42 MAPK rabbit monoclonal antibody (Cell Signaling, 1:1000) in 5% BSA (Bovine Serum Albumin, Sigma Aldrich) plus TBS/T buffer at 4°C overnight. HRP-conjugated secondary antibody was Goat anti-rabbit IgG antibody (Pierce, 1:10000) diluted in blocking solution. For analysis of phosphorylated eukaryotic Initiation Factor α (eIF2α-P), the membrane was incubated with anti-phospho-eIF2α polyclonal antibody (Invitrogen, 1:1000) in blocking solution at 4°C overnight. Membranes were stripped and reprobed with a rabbit polyclonal antibody that recognizes both the phosphorylated and unphosphorylated forms of eIF2α (eIF2α)(kindly provided by Dr. Thomas E. Dever). HRP-conjugated secondary antibody was Goat anti-rabbit IgG antibody (Pierce, 1:10000) diluted in blocking solution. Membranes were also probed with a mouse monoclonal antibody against Phosphoglycerate kinase (Pgk1p) (Molecular Probes, Invitrogen, 1:500) as a loading control.
Proteins were detected using a chemiluminescent substrate (SuperSignal West Pico, Thermo Scientific), and membranes were exposed to X-ray film, which were then scanned with a Molecular Imager FX Pro Plus (Bio-Rad, Hercules, CA). Digital image intensity was quantified using Quantity One 4.5.2 software (BioRad). Protein bands were quantified according to the ratio of the intensity of the test protein relative to the intensity of its Pgk1p loading control. The obtained values were averaged from duplicate experiments. Quantitative units were expressed as CNT*mm2 or Contour Quantity. This is described as the sum of the intensities of all the pixels within the band boundary multiplied by the area of each pixel (Quantity One, Bio-Rad). Error bars represent the Standard Error of the mean (STDError mean), calculated as the standard deviation (STDEV)/Square root (SQRT) of the count.
Alkaline phosphatase (PPase) treatment of protein extracts
To generate dephosphorylated proteins, 50 μg of whole yeast cell protein extract were incubated with 50U (1U/μg) of Calf Intestinal Alkaline Phosphatase (CIP or PPase, New England Biolabs) in the presence of 1X CIP buffer (10X NEB 3, New England Biolabs) and 5X Protease Inhibitors cocktail, EDTA Free (50X stock, Roche) for 30 min at 37°C. Samples were denatured at 95°C for 5 min and subjected to SDS-PAGE and Western blot analysis.
Wt, myo1Δ, tor1 Δ, myo1Δtor1 Δ, wsc1 Δ and myo1 Δwsc1 Δ strains were grown to OD600 between 0.5–0.8 AU at 26°C with continuous shaking at 226 rpm. 5uL of serial dilutions ranging from 1x107–1x102 cells/mL were spotted onto CSM or selection media agar plates containing 2% Glucose and 1X Nitrogen base. Plates were incubated at 26°C to observe growth after three days of incubation. Strains expressing the temperature sensitive tor2-21 ts mutation were streaked on CSM or selection media agar plates, and were incubated at 26°C and 37°C for 2.5 days.
The authors thank Drs. Brian C. Rymond, Estela Jacinto, Thomas E. Dever, and Michael N. Hall for their kind contribution of essential reagents and yeast strains. We also thank Sahily González-Crespo and Lilliam Villanueva-Alicea for their excellent technical support. This work was supported by a SCORE Award number (5-SC1AI081658-04) from the National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of General Medical Sciences (NIGMS). Partial support for this project was provided through Awards by RCMI (G12RR-03051-26) & (8 G12-MD007600) and MBRS-RISE (R25GM061838).
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