Nuclear export of tRNA is an essential eukaryotic function, yet the one known yeast tRNA nuclear exporter, Los1, is nonessential. Moreover recent studies have shown that tRNAs can move retrograde from the cytosol to the nucleus by an undefined process. Therefore, additional gene products involved in tRNA nucleus–cytosol dynamics have yet to be identified. Synthetic genetic array (SGA) analysis was employed to identify proteins involved in Los1-independent tRNA transport and in regulating tRNA nucleus–cytosol distribution. These studies uncovered synthetic interactions between los1 and pho88 involved in inorganic phopshate uptake. Further analysis revealed that inorganic phosphate deprivation causes transient, temperature-dependent nuclear accumulation of mature cytoplasmic tRNA within nuclei via a Mtr10- and retrograde-dependent pathway, providing a novel connection between tRNA subcellular dynamics and phosphate availability.

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TRANSPORT of tRNA from its nuclear site of biogenesis to the cytosol is an essential eukaryotic process as tRNA must be available for translation, which occurs in the cytosol. Intracellular tRNA movement in the budding yeast Saccharomyces cerevisiae involves several steps. First, end-matured pre-tRNAs are transported from nucleus to cytosol ("primary tRNA nuclear export"). Once in the cytosol, pre-tRNAs containing introns are spliced by the splicing endonuclease complex located on the cytosolic surface of the mitochondrial outer membrane (YOSHIHISA et al. 2003). Numerous nucleoside modification steps also occur in the nucleus and cytosol (HOPPER and PHIZICKY 2003). Mature cytosolic tRNA are then available for translation. Recent work demonstrated that mature tRNA can move from cytosol to nucleus (SHAHEEN and HOPPER 2005; TAKANO et al. 2005) and vice versa ("re-export"; WHITNEY et al. 2007). However, many of the gene products involved in the tRNA nucleus-to-cytosol and cytosol-to-nucleus transport pathways have yet to be defined.

Transport of many macromolecules between the nucleus and the cytosol requires the small GTPase, Ran, and its protein-binding partners, the ß-importins. Ran is maintained in the GTP-bound state in nuclei by Ran guanosine exchange factor (RanGEF), which is encoded in yeast by PRP20 and resides in the nucleus. ß-Importin family members that export cargo from the nucleus to the cytosol bind their cargo only in the presence of RanGTP and then mediate interactions with the nuclear pore complexes to allow the entire complex to move to the cytosol. Once in the cytosol, RanGTP interacts with the Ran GTPase activating protein (RanGAP), encoded by RNA1, which activates GTP hydrolysis, leading to conformational changes that cause the heterotrimeric complexes to dissociate, releasing the cargo in the cytosol (CORBETT et al. 1995; GORLICH and KUTAY 1999; WEIS 2003). For tRNA nuclear export, Los1, a member of the ß-importin family, binds tRNA in a RanGTP-dependent manner (HELLMUTH et al. 1998). The mammalian homolog of Los1, Exportin-t (Xpo-t), has also been shown to bind end-processed tRNA, with or without introns, as part of a heterotrimeric complex with RanGTP (ARTS et al. 1998a,b; KUTAY et al. 1998).

Inhibiting the Los1-dependent tRNA transport pathway through a temperature-sensitive mutation of RNA1 (rna1-1) or mutation of LOS1 caused nuclear accumulation of tRNA (SARKAR and HOPPER 1998). Similarly, blocking Xpo-t-mediated tRNA transport through microinjection of Xpo-t antibodies in Xenopus oocytes reduced nuclear export of tRNA^Phe by 99% (ARTS et al. 1998b). Blocking transport through mutation of RNA1, PRP20, LOS1, or some nucleopore genes also caused accumulation of intron-containing pre-tRNA (HOPPER et al. 1978, 1980; KADOWAKI et al. 1993; SHARMA et al. 1996), which indicated that these proteins were involved in the transport of end-matured pre-tRNA to the cytosol.

Numerous mutations have been identified that affect tRNA nucleus–cytosol distribution without causing accumulation of intron-containing pre-tRNA. These include temperature-sensitive mutations of methionyl- (mes1-1), isoleucyl- (ils1-1), and tyrosyl- (tys1-1) aminoacyl-tRNA synthetases (SARKAR et al. 1999), 3' tRNA CCA-nucleotidyl transferase (cca1-1; GROSSHANS et al. 2000; FENG and HOPPER 2002), and the tef1 tef2-1 double mutation of the eukaryotic elongation factor 1A (GROSSHANS et al. 2000). Lack of intron-containing pre-tRNA accumulation indicates that tRNA nuclear accumulation observed in these mutants occurs via accrual of fully processed, mature tRNA from the cytosol, and not by inhibition of the primary tRNA nuclear export pathway that exports pre-tRNA to the cytosol.

One genetic approach for defining parallel pathways that accomplish essential cellular functions is the identification of new mutations that have synthetic interactions with known mutations that impair or block one of the pathways. As tRNA nuclear export is an essential process and LOS1 is a known nonessential nuclear exporter of tRNA (HURT et al. 1987), this approach should be useful for the identification of genes that encode proteins participating in Los1-independent transport of end-matured pre-tRNA and mature tRNA and, possibly, negative regulators of tRNA retrograde transport.

Previous work employing standard synthetic lethal methodology identified several synthetic interactions with los1. The genes identified affected a variety of cellular processes, including tRNA aminoacylation (SIMOS et al. 1996a), tRNA modification (SIMOS et al. 1996a), Pol III transcription (SIMOS et al. 1996b), and translation (HELLMUTH et al. 1998; GROSSHANS et al. 2000). Synthetic genetic array (SGA) analysis (TONG et al. 2001) allows extensive, large-scale, systematic searches for genetic interactions. This approach was utilized with a collection of promoter replacement alleles of essential genes and los1 (DAVIERWALA et al. 2005). Depletion of Taf3, a subunit of the Pol II transcription initiation complex, and depletion of Pop5, a subunit of both RNase MRP (pre-rRNA cleavage) and RNase P (pre-tRNA 5'-end processing), were found to have synthetic interactions with los1 (DAVIERWALA et al. 2005). None of the previously reported studies have identified tRNA nuclear exporters that function in parallel to Los1 or regulators of tRNA nucleus–cytosol distribution.

Here, we describe the results of SGA screens of los1 using the yeast MATa deletion collection. Novel synthetic interactions were observed between los1 and snt309, aro7, rpl12a, rpl13b, lrp1, gtr1, gtr2, or pho88. Of these verified candidates, gtr1, gtr2, and pho88 caused tRNA nuclear accumulation. As gtr1, gtr2, and pho88 have been previously shown to influence inorganic phosphate (P_i) uptake (BUN-YA et al. 1992; YOMPAKDEE et al. 1996; LAGERSTEDT et al. 2005), we examined the affect of P_i starvation upon tRNA nucleus–cytosol distribution. Transient, temperature-dependent, tRNA nuclear accumulation was observed in wild-type cells grown on synthetic media lacking P_i for 1–2 hr at 23° or 30°. Northern and heterokaryon analyses of P_i-deprived cells and of pho88 cells indicated that the observed nuclear accumulation was due to accrual of mature tRNA, which entered the nucleus via retrograde transport. This work provides a novel connection between P_i availability and tRNA nucleus–cytosol distribution.

Strains and media:

Strains:

Yeast strains used for these studies are listed in Table 1. All plasmid transformations were made by lithium acetate (LiOAc) transformation (SCHIESTL and GIETZ 1989). Deletions were accomplished by LiOAc transformation of the parent yeast strain with a PCR product cassette that replaced the endogenous gene. Sequences for the oligonucleotides used are listed in supplemental Table S1 at http://www.genetics.org/supplemental/. The los1::natMX4 cassette was generated by PCR amplification using oligonucleotide primers RLH007A and RLH007B and a natR-MX4 template (p4339; TONG et al. 2001). LOS1KO1B was derived from the transformation of Y3656 with the los1::natMX4 cassette. The deletion of LOS1 was verified using the primers RLH010A and RLH010B. The pho88::natMX4 cassette was generated by PCR amplification using oligonucleotide primers RLH017A and RLH017B and a natR-MX4 template (GOLDSTEIN and MCCUSKER 1999). 8MS88N2C was derived from the transformation of MS739 with the pho88::natMX4 cassette. BY88F1 was derived from the transformation of BY4741 with the pho88::natMX4 cassette. The pho88::hphMX4 cassette was generated by PCR amplification using the oligonucleotide primers RLH017A and RLH017B and the hphR-MX4 template (GOLDSTEIN and MCCUSKER 1999). BY88D1 and BY88G4 were derived from independent transformations of BY4741 with the pho88::hphMX4 cassette. 8MS88H2D was derived from the transformation of MS739 with the pho88::hphMX4 cassette. YD05055 (los1::kanMX) was transformed with YCpLOS1 (HURT et al. 1987) and then with the pho88::hphMX4 cassette to form strain los1 pho88 + YCpLOS1. The deletion of PHO88 was verified using primers RLH017C, RLH017D, and RLH017E. The strain los1 pho88 + YCpLOS1 was grown on nonselective media to allow plasmid loss from which los1 pho88 was derived.

View this table: In this window In a new window   TABLE 1 Yeast strains employed

 

Media:

Yeast strains were maintained on YEPD [yeast extract, trypticase peptone, dextrose, supplemented with adenine (0.04 g/liter), uracil (0.04 g/liter)] medium; SC (synthetic complete) defined medium; Difco yeast nitrogen base without amino acids (6.7 g/liter; Becton Dickinson, Sparks, MD) supplemented with amino acids, dextrose (2%), adenine (0.04 g/liter), and uracil (0.04 g/liter). SC–P_i liquid medium is synthetic defined medium lacking K₂PO₄ [the source of P_i in SC; yeast nitrogen base lacking amino acids and K₂PO₄ (5.7 g/liter; ForMedium, Norwich, UK) supplemented with amino acids, uracil, and 1.0 g/liter KCl]. Solid YEPD medium supplemented with 2 g/liter KH₂PO₄ (Fisher) was utilized for the "wild-type" heterokaryon fluorescence in situ hybridization (FISH) analysis. To select for natMX cassette integration, the strains were grown on YEPD + clonNAT (100 mg/liter; Werner BioAgents, Jena, Germany) solid medium. To select for hphMX cassette integration, the strains were grown on YEPD + hygromycin B (300 mg/liter; Calbiochem, La Jolla, CA) solid medium.

Genetic analysis:

SGA was performed and scored as previously described (TONG et al. 2001) using LOS1KO1B as the bait strain. The final replica plates were grown at 22° or 37°. Tetrad dissection was done by standard procedures.

Growth assay:

Serial dilutions were made of the indicated yeast cultures. Aliquots (5 µl) of each dilution were spotted onto each plate of the indicated solid medium. The plates were incubated for 2–3 days at the indicated temperature. Images of the plates were captured using Eagle Eye II (Stratagene, La Jolla, CA), digital camera, or scanner (ScanMaker 8700 by Microtek, Carson, CA). Adobe Photoshop 5.0 was used for image assembly.

Fluorescence in situ hybridization:

FISH was performed as previously described (SARKAR and HOPPER 1998) with the modifications detailed in STANFORD et al. (2004). Heterokaryons for FISH were mated and grown as described in SHAHEEN and HOPPER (2005) except the solid YEPD medium used for the "wild-type" mating was medium supplemented with 2 g/liter KH₂PO₄ (Fisher) to prevent premature induction of the PHO pathway, as low levels of PHO5 expression have been observed when cells are grown in YEPD medium (YOSHIDA et al. 1989a,b). Oligonucleotides used as probes are listed in supplemental Table S1 at http://www.genetics.org/supplemental/. Each slide contained positive and negative controls for tRNA nuclear accumulation. When adjectives describing the relative amount of tRNA nuclear accumulation are used, the probes were hybridized under the same conditions. All critical experiments were independently viewed and scored by at least two people, one of whom was unaware of the experimental details. A Nikon Microphot-FX microscope was used to observe fluorescence and a Sensys charge-coupled device camera (Photometrics, Tucson AZ) using QED software (QED Imaging, Pittsburgh) was used to capture the images. Adobe Photoshop 5.0 was used for image assembly.

Northern analysis:

Small RNAs were extracted from yeast cultures grown to densities similar to those used for FISH as previously described (HOPPER et al. 1980). Samples (10 µg of RNA) were electrophoretically separated at 4° in 10% polyacrylamide gel containing 8 M urea and 1x TBE (0.09 M Tris, 0.09 M borate, 0.001 M ethylenediaminetetraacetic acid). RNAs were electrophoretically transferred onto Hybond N+ membranes (Amersham Pharmacia) using a Hoefer TE42 Transphor apparatus (Hoefer Scientific) filled with 1x TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA). Membranes were prehybridized at 37° in 4x SSC (1x is 0.15 M NaCl, 0.015 M sodium citrate), 2.5x Denhart's solution [1x is 0.02% Ficoll (type 400), 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin], 50 µg/ml single-stranded salmon sperm DNA, and 0.05% sodium dodecyl sulfate. Probe hybridization was at 37° in the same buffer with the addition of oligonucleotide probes (supplemental Table S1 at http://www.genetics.org/supplemental/) that were 5' end labeled with [³²P]ATP using T4 polynucleotide kinases (Promega, Madison, WI). After hybridization, membranes were UV crosslinked, washed in 2x SSC, and used to expose Kodak BioMax MS film (Eastman Kodak, Rochester, NY).

Acid phosphatase assay:

Acid phoshatase activity was determined as previously described (BYRNE et al. 2004) except that yeast cultures were grown to early log phase and the volume of cells was increased to compensate for the reduced number of cells.

SGA analysis of los1 uncovers eight novel interactions:

SGA was performed twice using LOS1KO1B (los1::nat^R) as bait. All of the incubation steps for the first screen were conducted at 22°, the standard temperature for SGA. The final replica plates of the second screen were incubated at 37°, a temperature that exaggerates the los1 phenotype in some strains (HOPPER et al. 1980). The first screen provided 18 novel candidate interactions, whereas the second screen uncovered 118 candidates. As only six candidates appeared in both screens—deletions of ARC1 (YGL105W), LDB16 (YCL005W), LRP1 (YHR081W), RCY1 (YJL204C), WHI3 (YNL197C), and RPL13B (YMR142C)—the total number of candidates was 131 (supplemental Table S2 at http://www.genetics.org/supplemental/). Candidates of the first screen did not necessarily overlap with the candidates found in the second screen, possibly due to changes in the phenotype of individual deletion strains at different temperatures, manipulation errors, false positives, or false negatives. ARC1, a known synthetic lethal interaction (SIMOS et al. 1996b), was found in both screens, indicating that the assay was functioning as predicted.

Tetrad analysis and growth assays of selected haploid progeny were utilized to detect and verify synthetic lethal, slow, or enhanced growth interactions. Tetrad analysis of 97 candidate interactions revealed eight bona fide los1 interactions (Table 2); only three of these appeared as candidates in both searches. Synthetic enhanced growth interaction was observed between los1 and snt309 (YPR101W). Synthetic slow-growth interactions were observed between los1 and aro7 (YPR060C), rpl12a (YEL054C), rpl13b (YMR142C), lrp1 (YHR081W), gtr1 (YML121W), or gtr2 (YGR163W).

View this table: In this window In a new window   TABLE 2 Bona fide los1 interactions

 
A synthetic lethal interaction between los1 and pho88 (YBR106W) was documented upon tetrad dissection at 23° by the absence of nonparental ditypes and the absence of los1 pho88 containing progeny in tetratype asci as predicted by two-gene synthetic lethality (Figure 1A). However, we were able construct a viable los1 pho88 strain via direct mutagenesis (see MATERIALS AND METHODS). The los1 pho88 strain exhibited slower growth at 23° than strains containing either individual mutation (Figure 1B). The slow growth vs. synthetic lethal interactions observed between los1 and pho88 are addressed in the DISCUSSION.

View larger version (56K): In this window In a new window Download PPT slide   FIGURE 1.— Analysis of spore viability and growth of cells with PHO88 and/or LOS1 deletions. (A) Tetrad dissection of los1 (LOS1KO1B) x pho88 (pho88MATa) and of los1 (LOS1KO1B) x npr1 (npr1MATa) on YEPD. (B) BY4741, YD05055 (los1 MATa), los1pho88, and pho88 MATa were grown to saturation, and then equal amounts of cells from each strain were used to make two series of dilutions. Aliquots (5 µl) of each dilution were placed on solid synthetic complete medium. Plates were incubated for 2 days at the indicated temperatures.

 

SGA analysis uncovered genes influencing tRNA nucleus–cytosol distribution:

The subcellular distributions of tRNA between the nucleus and the cytosol of selected synthetic interactors were evaluated using fluorescence in situ hybridization (SARKAR and HOPPER 1998). Gene deletions that had synthetic slow-growth phenotypes were expected to cause tRNA nuclear accumulation if the growth defect was due to impairment of tRNA export. Nuclear accumulation of both tRNA^Met and tRNA^Tyr, as indicated by the increased FITC signal that colocalizes with the DAPI-stained DNA, was observed in los1 (Figure 2A, column 2), pho88 (Figure 2A, column 5), gtr1 (supplemental Figure S1 at http://www.genetics.org/supplemental/ and data not shown), and gtr2 (supplemental Figure S1 at http://www.genetics.org/supplemental/ and data not shown) strains (Table 2). Several independent pho88 strains were generated, and all exhibited tRNA nuclear accumulation (Figure 2B, columns 2 and 4; also see supplemental Figure S1 at http://www.genetics.org/supplemental/). In contrast, arc1 cells exhibited nuclear accumulation of tRNA^Met while the cellular distribution of tRNA^Tyr was similar to wild-type cells (Figure 2A, column 3). Deletion of arc1 influencing the nucleus–cytosol distribution of tRNA^Met, but not tRNA^Tyr, is consistent with the function of Arc1 as a cofactor of the methionyl- and glutamyl-tRNA synthetases (SIMOS et al. 1996a). However, not all deletions that had synthetic slow-growth interactions with los1 caused tRNA nuclear accumulation. For example, wild-type, rpl13b, and rpl12a cells had a similar cellular distribution of tRNA^Met and tRNA^Tyr (Figure 2A, columns 1 and 4, and supplemental Figure S1 at http://www.genetics.org/supplemental/, respectively; Table 2).

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 2.— Subcellular distribution of tRNA in selected SGA candidate deletions. (A) Location of tRNA within candidate deletion strains identified by SGA was determined by FISH analysis of BY4741 (wild type), YD05055 (los1), arc1MATa, rpl13bMATa, and pho88MATa cells. (Row 1) tRNAMet; (row 3) tRNATyr; (rows 2 and 4) DAPI stain of row 1 and 3 cells, respectively. (B) FISH analysis of BY4741 (wild type), BY88D1 (pho88::hphR in BY4741), MS739 (kar1-1), and 8MS88N2C (pho88::natR in MS739) cells. (Row 1) tRNAMet. (Row 3) tRNATyr. (Rows 2 and 4) DAPI stain of row 1 and row 3 cells, respectively.

 
Candidate deletions that caused tRNA nuclear accumulation had been implicated in uptake of P_i. Gtr1 and Gtr2 formed a small GTPase complex (NAKASHIMA et al. 1999) that negatively regulated the Ran cycle (NAKASHIMA et al. 1996) and was required for efficient P_i uptake in response to P_i starvation (BUN-YA et al. 1992; LAGERSTEDT et al. 2005). Deletion of PHO88 caused impaired P_i uptake and slowed growth (YOMPAKDEE et al. 1996). PHO88 was selected for further study because it had strong synthetic interactions with los1, its deletion caused tRNA nuclear accumulation, and it has been shown to impact the regulation of the PHO pathway (YOMPAKDEE et al. 1996).

P_i deprivation of wild-type cells caused tRNA nuclear accumulation:

We obtained a plasmid encoding an N-terminal GST-tagged Pho88 (MARTZEN et al. 1999) and found that GST-Pho88 was located in the endoplasmic reticulum (ER; data not shown). This location is consistent with the results of the genomewide protein localization study that employed C-terminal GFP tags (HUH et al. 2003) and the observed membrane association of Pho88 (YOMPAKDEE et al. 1996). As Pho88 was located in the ER, it was unlikely to directly bind and transport tRNA from the nucleus to the cytosol. Given that deletion of PHO88 caused a defect in P_i uptake from media (YOMPAKDEE et al. 1996), it was possible that the low intracellular P_i levels caused the tRNA nuclear accumulation. This possibility had precedent as amino acid starvation has been shown to cause tRNA nuclear accumulation (SHAHEEN and HOPPER 2005). If pho88 cells redistributed tRNA in response to low intracellular P_i levels, then wild-type cells that cannot import P_i due to lack of substrate should also have tRNA nuclear accumulation.

To address this prediction, wild-type cells (strain BY4742) were grown on synthetic complete media lacking KH₂PO₄ (SC–P_i) for various lengths of time. This approach allowed a controlled starvation period and minimized the risk of suppressor appearance. The cellular distributions of tRNA were then determined by FISH. As expected, tRNA^His was evenly distributed in wild-type cells grown in fresh SC media for 2 hr (Figure 3A, column 1). An even distribution of tRNA^His was also visible in cells grown for 30 min in SC–P_i (data not shown). Cells grown at 23° in SC–P_i for 1 hr displayed nuclear accumulation of tRNA^His (Figure 3A, column 2, row 1) as did cells grown at 30° in SC–P_i for 1 hr (Figure 3A, column 2, row 3). Thus, cells redistributed tRNA in response to P_i removal.

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 3.— tRNA location within Pi-deprived cells. (A) Pi starvation time course of wild-type cells grown at 23°, 30°, and 37°. BY4742 cells grown in SC (column 1) or SC–Pi (columns 2–4) for the indicated amount of time at 23° (rows 1 and 2), 30° (rows 3 and 4), or 37° (rows 5 and 6) were analyzed by FISH. The cellular distribution of tRNAHis is shown (rows 1, 3, and 5). The same cells were stained with DAPI (rows 2, 4, and 6). (B) Pi-starved pho4 cells and YEPD-grown pho88 pho4 and pho88 cells. Cellular distributions of tRNATyr (row 1) determined by FISH, of pho4 MATa cells grown in fresh SC or SC–Pi for 1.5 hr, and of pho88 MATa and pho4 pho88. Cells were stained with DAPI to reveal the location of DNA (row 2).

 
Unexpectedly, tRNA nuclear accumulation in response to P_i deprivation demonstrated temperature- and time-dependent components. While FISH is primarily a qualitative technique, the tRNA^His nuclear accumulation in cells grown at 23° reproducibly appeared to be more intense than in cells grown at 30° (Figure 3A, columns 2 and 3, rows 1 and 3). In contrast, cells grown at 37° in SC–P_i did not exhibit nuclear accumulation of tRNA^His (Figure 3A, columns 1–4, row 5). When cells were grown at 23° or 30° for 2 hr in SC–P_i, tRNA nuclear accumulation appeared diminished in comparison to the 1-hr time point. Moreover, cells grown for 3.5 or 12 hr in SC–P_i had tRNA^His nucleus–cytosol distributions similar to cells grown in fresh SC. The redistribution of tRNA^Tyr and tRNA^Met in response to growth in SC–P_i mirrored the demonstrated redistribution of tRNA^His (data not shown). The data indicated that denying wild-type cells readily available sources of P_i caused temperature-dependent, transient tRNA nuclear accumulation, in contrast to pho88 cells, which constitutively accumulated tRNA in the nucleus.

Activation of the PHO pathway is a well-studied response to P_i deprivation (CARROLL and O'SHEA 2002). The subcellular location and activity of Pho4p, the master transcription factor of the P_i starvation response, is regulated through phosphorylation at four sites. Pho4 is cytosolic, inactive, and highly phosphorylated when cells are grown in media rich in P_i as the Pho80/Pho85 cyclin–CDK complex is fully active and maintains Pho4 in a highly phosphorlyated state (CARROLL and O'SHEA 2002). When cells are grown in low P_i medium, the Pho80/Pho85 cyclin–CDK complex is partially inhibited by Pho81, causing Pho4 to be only partially phosphorylated. In this partially phosphorylated state, Pho4 is nuclear and activates the transcription of genes such as Pho84, a high-affinity P_i transporter (50% of maximal expression). Partially phosphorylated Pho4 has a minimal effect upon the transcription of other phosphate-responsive genes, including the acid phosphatase (rAPase), encoded by PHO5, which is expressed at 10% of maximum levels (SPRINGER et al. 2003). Full inhibition of Pho80/Pho85 by Pho81 in response to P_i deprivation causes Pho4 to become fully dephosphorylated and competent in activating transcription of all its target genes, including Pho5 (SPRINGER et al. 2003).

To ascertain when tRNA nuclear accumulation occurs with respect to the PHO pathway-mediated gene expression, we determined the enzymatic activity of secreted Pho5. As the amount of the p-nitrophenylphosphate converted to p-nitrophenolate is proportional to the amount of Pho5 (TOH-E et al. 1973), determining production of p-nitrophenolate provides an indirect measurement of Pho5 expression. The rAPase activity was assayed (BYRNE et al. 2004) using intact, early log-phase wild-type cells (strain BY4742) grown in SC–P_i, which duplicated the growth conditions of 23° cell cultures used for FISH analysis. Cells grown for 2 hr in SC–P_i had 145% of basal activity, whereas cells grown for 3.5 hr in SC–P_i had 278% of basal activity (Table 3). After 10 hr of growth in SC–P_i, cells had 1100% of basal activity (Table 3). The rAPase activity indicates that Pho5 has low levels of expression at the time points when tRNA nuclear accumulation occurs (1–2 hr of P_i deprivation).

View this table: In this window In a new window   TABLE 3 Acid phosphatase activity

 
Nuclear accumulation of tRNA appears to be an early response to P_i deprivation as it occurs slightly after Pho4p translocation from the cytosol to the nucleus (KAFFMAN et al. 1998) in contrast to the later, strong induction of Pho5p expression (Table 3). To address the possibility that the tRNA nuclear accumulation in response to P_i deprivation could be dependent on the PHO pathway, the subcellular tRNA distributions in P_i-deprived and replete pho4 or pho81 cells were determined. Unstarved pho4 cells grown at 23° in fresh SC have a slight accumulation of tRNA^Tyr (Figure 3B, column 1). As pho4 and pho81 cells grown at 23° in SC–P_i for 1.5 hr also exhibited nuclear accumulation of tRNA (Figure 3B, columns 2 and data not shown), an intact PHO pathway is not required for tRNA nuclear accumulation induced by P_i deprivation. In addition, pho4 pho88 cells displayed tRNA nuclear accumulation that is similar to the accumulation within pho88 cells (Figure 3B, columns 4 and 3, respectively). Therefore, P_i deprivation-induced tRNA nuclear accumulation is an early, signal-mediated response to P_i deprivation that does not require the PHO pathway.

Mature tRNA accumulated within nuclei in response to P_i deprivation enter via the retrograde transport pathway:

The current model of tRNA nucleus–cytosol transport has two modes for tRNA nuclear egress: primary nuclear export of newly synthesized tRNAs and re-export of mature tRNA. Blocking the primary nuclear export of tRNAs by mutation of LOS1, or any other members of the Ran pathway, causes accumulation of intron-containing pre-tRNAs. If P_i deprivation or deletion of PHO88 caused a block in the primary tRNA nuclear export pathway, then accumulation of intron-containing pre-tRNAs should have occurred. To determine whether tRNA nuclear accumulation observed in response to P_i deprivation and in pho88 cells occurs due to an inhibition of the primary export pathway or the re-export pathway, tRNA from wild-type cells grown in SC–P_i and pho88 cells were analyzed by Northern blot analysis. Depriving wild-type cells of P_i did not cause accumulation of intron-containing pre-tRNAs (Figure 4A). The independently derived pho88 strains did not exhibit accumulation of intron-containing pre-tRNAs (Figure 4). Therefore, mature tRNA had accumulated within nuclei of pho88 cells and P_i-deprived wild-type cells.

View larger version (80K): In this window In a new window Download PPT slide   FIGURE 4.— Northern analysis of Pi-starved wild-type cells and pho88 deletion strains. (A) Samples (10 µg) of small RNAs from BY4741 (wild-type) cells grown in SC (column 1) or in SC–Pi for the amount of time indicated (columns 2–5) at 23° from pho88 MATa cells (column 6) and from YD05055 (los1) cells (column 7) were separated by electrophoresis and then transferred to a membrane. The membrane was probed with 32P-labeled oligonucleotides complementary to tRNAIle and detected by autoradiography. Arrows indicate intron-containing pre-tRNAs. Dashes indicate mature tRNAs. (B, top) Samples (10 µg) of small RNAs from BY4742 (wild-type) cells, from YD05055 (los1) cells, and from cells of three independently derived pho88 strains (BY88D1, BY88F1, BY88G4) grown in SC at 23° were separated by electrophoresis and then transferred to a membrane. The membrane was probed with 32P-labeled oligonucleotides complementary to tRNATyr and detected by autoradiography. Arrow indicates intron-containing pre-tRNAs. Dashes indicate mature tRNAs. (Bottom) Photo of the ethidium–bromide-stained polyacrylamide gel, prior to transfer, illuminated by ultraviolet light.

 
Since Northern analysis indicated that the tRNA nuclear accumulation observed in response to P_i deprivation was not due to impaired export of newly synthesized tRNA, tRNA nuclear accumulation was likely to have occurred through the tRNA retrograde pathway, similar to amino-acid-starvation-induced tRNA redistribution (SHAHEEN and HOPPER 2005). To verify that tRNA retrograde nuclear accumulation occurs during P_i deprivation and when Pho88 is absent, the distribution of tRNA^Glu-D was determined in "wild-type" heterokaryons grown in SC–P_i and in heterokaryons without Pho88. Heterokaryons were generated by mating yeast cells, in which one of the mating types carries a plasmid-expressing tRNA^Glu-D from Dictyostelium discoideum and a kar1-1 mutation (strains MS739 + tRNA^Glu-D or 8MS88H2D + tRNA^Glu-D), with KAR1 yeast cells (strain BY4741 or BY88D1). The kar1-1 mutation blocked nuclear fusion upon mating and resulted in cells with a shared cytosol and two separate nuclei, where only one nucleus carried the plasmid. If mature tRNA, in response to P_i deprivation, had entered nuclei through retrograde tRNA transport, then all nuclei in a heterokaryon would have accumulated tRNA^Glu-D when grown in SC–P_i. If it is independent of retrograde tRNA transport, then only nuclei that carried the plasmid-expressing tRNA^Glu-D should have accumulated tRNA^Glu-D when grown in SC–P_i.

Heterokaryons grown at 23° in SC–P_i for 1 or 2 hr, or in fresh SC for 2 hr, were subjected to FISH analysis. To quantify the data, heterokaryons were scored by determining if all, some, or none of the nuclei accumulate tRNA^Glu-D. Nearly every heterokaryon grown in SC had an even distribution of tRNA^Glu-D throughout the cell (Figure 5A, column 1; Table 4). Of the heterokaryons grown in SC–P_i for 1 hr, 83.5% accumulated tRNA^Glu-D in all nuclei (Figure 5A, column 2; Table 4), 14.4% accumulated tRNA^Glu-D in some nuclei, and 2.1% had no nuclear accumulation of tRNA^Glu-D. The majority of heterokaryons grown in SC–P_i for 2 hr still accumulated tRNA^Glu-D in all nuclei (72.7%; Figure 5A, column 3); however, as previously observed in haploid cells (Figure 3), the accumulation was not as prominent as the 1-hr time point and there was an increase in cells that have no nuclear accumulation (17.2 vs. 2.1%). The reduction of the total number of cells exhibiting tRNA nuclear accumulation after 2 hr of growth in SC–P_i was expected because the tRNA nucleus–cytosol distribution of wild-type cells returned to an even distribution between 2 and 3.5 hr of growth in SC–P_i. Since tRNA^Glu-D accumulates in all of the nuclei of the majority of P_i-deprived "wild-type" heterokaryons, similar to amino-acid-deprived "wild-type" heterokaryons (SHAHEEN and HOPPER 2005), the tRNA that accumulated in response to P_i deprivation entered the nucleus via retrograde tRNA transport of cytosolic tRNA.

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 5.— Retrograde transport in pho88 and Pi-deprived wild-type cells. (A) "Wild-type" heterokaryon cells grown in fresh SC (column 1), SC–Pi for 1 hr (column 2) or SC–Pi for 2 hr (column 3), and "88" heterokaryons (column 4) were analyzed by FISH. (Rows 1 and 2) tRNAGlu-D. (Rows 2 and 4) DAPI stain. (B) mtr10 MATa cells grown in fresh SC or SC–Pi for 1.5 hr at 23° were analyzed by FISH. (Row 1) tRNAHis. (Row 2) DAPI stain.

 

View this table: In this window In a new window   TABLE 4 Heterokaryon assay of tRNA retrograde movement

 
The anticipated cause of tRNA nuclear accumulation within pho88 cells was low intracellular levels of P_i caused by defective P_i uptake. Therefore, the tRNA accumulated within the nuclei of pho88 cells was also expected to have entered the nucleus through retrograde transport. Heterokaryons homozygous for pho88 ("88") were subjected to FISH analysis. Consistent with our prediction, 95.2% of "88" heterokaryons accumulated tRNA^Glu-D in all nuclei (Figure 5A, column 5; Table 4). The data indicate that the tRNA accumulated within the nucleus of pho88 cells entered via retrograde tRNA transport.

Amino-acid-starvation-induced tRNA nuclear accumulation requires the ß-importin, Mtr10, as mtr10 cells exhibit an even distribution of tRNA when grown in SC lacking amino acids (SHAHEEN and HOPPER 2005). If Mtr10 is necessary for P_i-deprivation-induced tRNA nuclear accumulation, then P_i-deprived mtr10 cells will exhibit an even distribution of tRNA. When grown in SC–P_i at 23° for 1.5 hr, mtr10 cells showed the same tRNA nucleus–cytosol distribution as mtr10 cells grown in SC (Figure 5B, columns 2 and 1, respectively). The data indicated that P_i-deprivation-induced tRNA nuclear accumulation was Mtr10 dependent and therefore occurred by a mechanism shared with amino-acid-starvation-induced tRNA nuclear accumulation.

We undertook these studies to systematically query nonessential yeast genes for synthetic interactions with los1 to learn more about tRNA subcellular dynamics and we discovered a role for the availability of P_i in this process. SGA technology uncovered 131 candidate interactions with los1. While 6 candidates were identified in both the 22° and the 37° assays, only 3 (lrp1, rpl13b, arc1) of these had bona fide interactions as determined by tetrad analysis and growth assays. Of the 97 candidates evaluated by tetrad dissection, eight novel interactions were uncovered (Table 2). The SGA false-positive rate for los1 is 90.7% compared to the 25–50% success rate reported for other baits (TONG et al. 2004). The false-positive rate of the candidates that appeared in both screens (3 of 6) is 50%. This indicates that many false positives could have been eliminated by repeating the assays at both 22° and 37°. However, we note that PHO88 would not have been analyzed if SGA had been performed only at 37°. Since we previously noted that amino acid starvation causes tRNA nuclear accumulation (SHAHEEN and HOPPER 2005), one may have expected to uncover SHR3, a likely equivalent to Pho88 for amino acid permeases, as a candidate. However, deletion of SHR3 was inviable by large-scale deletion (WINZELER et al. 1999) and was not included in the assay.

The eight novel synthetic interactions uncovered in this study affect amino acid synthesis, translation, mRNA and tRNA processing, and P_i uptake. The variety of cellular processes affected by the identified deletions and the data indicating that some of the identified deletions do not affect tRNA subcellular dynamics, raises the question as to why these synthetic interactions with los1 occur. RPL12A and RPL13B encode ribosomal proteins. The synthetic interactions between los1 and mutations of protein synthesis machinery, including tef2 and gcd11 (HELLMUTH et al. 1998; GROSSHANS et al. 2000), are likely the result of the cumulative impairment of translation by reduced availability of cytoplasmic tRNA (los1) and inefficient ribosomes. Aro7 is required for synthesis of tyrosine and phenylalanine. Deletion of ARO7 affects amino acid synthesis, and reduced amino acid availability may also affect the rate of protein synthesis. The genetic interaction between lrp1 and los1 may be caused by multiple defects in cellular RNA levels because previous studies uncovered synthetic lethal interactions between lrp1 and a variety of genes encoding defective tRNA, mRNA, and rRNA processing activities (HIERONYMUS et al. 2004; DAVIERWALA et al. 2005). Our studies uncovered a single synthetic-enhanced growth interaction between snt309 and los1. Since Snt309 is a component of spliceosomes (CHEN et al. 1999), it is unclear why snt309 and los1 would have enhanced genetic interactions.

Different synthetic interactions between los1 and pho88 were observed when SGA or direct mutagenesis was employed to generate double mutants. The cold-sensitive nature of the synthetic slow-growth interaction observed in los1 pho88 cells may explain why PHO88 was found as a SGA candidate when the array was performed at 22° but not at 37°. Tetrad dissection was performed at 23° on YEPD medium, which has low levels of P_i (YOSHIDA et al. 1989b). This may have contributed to the absence of los1 pho88 progeny. If low temperature and/or medium prevented the appearance of los1 pho88 colonies, then tetrad dissection of pho88 by los1 crosses on high-P_i medium at 30° may produce los1 pho88 colonies. Tetrad dissection also involves a starvation period to induce meiosis, whereas direct mutagenesis occurs on rich media. Thus, it is also possible that los1 pho88 spores are unable to germinate after this starvation.

Deletion of PHO88, GTR1, or GTR2 caused nuclear accumulation of mature tRNA. Simulating the P_i-uptake defect observed in pho88 cells by growing wild-type cells in SC–P_i caused transient, temperature-dependent tRNA nuclear accumulation without causing accumulation of intron-containing tRNAs. The data indicate that tRNA nuclear accumulation was not due to impaired export of newly synthesized tRNAs and that P_i-deprived wild-type and pho88 cells accumulated mature tRNA only in nuclei that entered through tRNA retrograde transport. Consistent with amino-acid-starvation-induced tRNA redistribution, the data also demonstrated that the tRNA nuclear accumulation observed in P_i-deprived cells was Mtr10 dependent.

Although we have demonstrated that the mature tRNA accumulating within nuclei of pho88 and P_i-deprived wild-type cells entered the nuclei via the retrograde transport pathway, the mechanism for the accumulation remains unclear. Accumulation could have occurred due to increased retrograde transport, blocked re-export, or simultaneous occurrence of both increased retrograde transport and blocked re-export. Mtr10 may function in a signal transduction pathway regulating retrograde transport and/or re-export or as part of a tRNA importing complex.

Nuclear accumulation of tRNA that occurred due to PHO88 deletion largely paralleled the accumulation that occurred in P_i-deprived wild-type cells. However, pho88 cells constitutively accumulated tRNA within nuclei, whereas the accumulation observed during P_i deprivation was transient in nature. This difference may be explained by the differences in how the cells were impaired for P_i uptake. In the absence of Pho88, cells were able to constitutively acquire very low levels of P_i, sufficient for slow growth (YOMPAKDEE et al. 1996). In contrast, P_i deprivation caused wild-type cells to undergo a two-phase response. During the first phase, when tRNA nuclear accumulation occurred, polyphosphate is mobilized (MOUILLON and PERSSON 2006), Pho84 is highly expressed, and Pho5 is slightly expressed. The second phase, when tRNA returned to normal distribution, was marked by increased expression of Pho5. Similarly, recent data demonstrated that acute glucose starvation causes tRNA nuclear accumulation, whereas an even distribution of tRNA occurred during prolonged starvation (WHITNEY et al. 2007). This indicates that cells may have acclimatized to prolonged starvation and redistributed previously accumulated tRNA via re-export. Cells with PHO88 deletions may never reach the extremely low levels of intracellular P_i required for this transition. As P_i-deprivation-induced tRNA nuclear accumulation was independent of Pho4 and Pho81, the signaling pathway(s) controlling tRNA distribution during the P_i deprivation time course have yet to be determined.

Although a goal of this work was to uncover tRNA export pathways that function in parallel to Los1 to export newly synthesized tRNA, such a pathway was not identified. This unidentified exporter(s) may have been missed by SGA or not included in the deletion collection, or it may be encoded by an essential gene that has yet to be recognized as a tRNA transporter. Instead, we successfully uncovered novel connections between los1 and mRNA processing (lrp1) and stable RNA processing (snt309) and additional connections to the ribosome (rpl13b, rpl12a) and amino acid synthesis (aro7). The novel synthetic interactions between los1 and gtr1, gtr2, or pho88 identified in this work provided a clear connection between tRNA subcellular dynamics and phosphate availability. Further studies are required to understand the signal transduction and the mechanism by which P_i levels regulate tRNA nucleus–cytosol distribution.

We thank H. Shaheen, M. Whitney, A. Murthi, and K. Stauffer for valuable scientific interactions. This work was supported by a fellowship to R.L.H. from the American Heart Association, by a grant to A.K.H. from the National Institutes of Health, and by grants to C.B. from the Canadian Institute of Health Research, Genome Canada, and Genome Ontario.

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