Inorganic phosphate deprivation causes tRNA nuclear accumulation via retrograde transport in Saccharomyces cerevisiae

Abstract

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 los1Delta and pho88Delta involved in inorganic phopsphate 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.

Figure 1.—

Analysis of spore viability and growth of cells with PHO88 and/or LOS1 deletions. (A) Tetrad dissection of los1Δ (LOS1KO1B) × pho88Δ (pho88ΔMATa) and of los1Δ (LOS1KO1B) × npr1Δ (npr1ΔMATa) on YEPD. (B) BY4741, YD05055 (los1Δ MATa), los1Δpho88Δ, and pho88Δ 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.

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 (los1Δ), arc1ΔMATa, rpl13bΔMATa, and pho88ΔMATa cells. (Row 1) tRNA^Met; (row 3) tRNA^Tyr; (rows 2 and 4) DAPI stain of row 1 and 3 cells, respectively. (B) FISH analysis of BY4741 (wild type), BY88ΔD1 (pho88∷hph^R in BY4741), MS739 (kar1-1), and 8MS88ΔN2C (pho88∷nat^R in MS739) cells. (Row 1) tRNA^Met. (Row 3) tRNA^Tyr. (Rows 2 and 4) DAPI stain of row 1 and row 3 cells, respectively.

Figure 3.—

tRNA location within P_i-deprived cells. (A) P_i starvation time course of wild-type cells grown at 23°, 30°, and 37°. BY4742 cells grown in SC (column 1) or SC–P_i (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 tRNA^His is shown (rows 1, 3, and 5). The same cells were stained with DAPI (rows 2, 4, and 6). (B) P_i-starved pho4Δ cells and YEPD-grown pho88Δ pho4Δ and pho88Δ cells. Cellular distributions of tRNA^Tyr (row 1) determined by FISH, of pho4Δ MATa cells grown in fresh SC or SC–Pi for 1.5 hr, and of pho88Δ MATa and pho4Δ pho88Δ. Cells were stained with DAPI to reveal the location of DNA (row 2).

Figure 4.—

Northern analysis of P_i-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–P_i for the amount of time indicated (columns 2–5) at 23° from pho88Δ MATa cells (column 6) and from YD05055 (los1Δ) cells (column 7) were separated by electrophoresis and then transferred to a membrane. The membrane was probed with ³²P-labeled oligonucleotides complementary to tRNA^Ile 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 (los1Δ) cells, and from cells of three independently derived pho88Δ strains (BY88ΔD1, BY88ΔF1, BY88ΔG4) 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 tRNA^Tyr 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.

Figure 5.—

Retrograde transport in pho88Δ and P_i-deprived wild-type cells. (A) “Wild-type” heterokaryon cells grown in fresh SC (column 1), SC–P_i for 1 hr (column 2) or SC–P_i for 2 hr (column 3), and “88Δ” heterokaryons (column 4) were analyzed by FISH. (Rows 1 and 2) tRNA^Glu-D. (Rows 2 and 4) DAPI stain. (B) mtr10Δ MATa cells grown in fresh SC or SC–P_i for 1.5 hr at 23° were analyzed by FISH. (Row 1) tRNA^His. (Row 2) DAPI stain.