Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

Abstract

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.

FIG. 1.

eIF6 is phosphorylated in HeLa and yeast cells. (A) Cell extracts of HeLa cells were fractionated into nuclear (N) and cytoplasmic (C) fractions. Where indicated, aliquots of each fraction were preincubated with calf intestinal alkaline phosphatase (CIAP). These fractions (lanes d and e) and untreated controls (lanes b and c) along with purified rabbit reticulocyte eIF6 (Retic. eIF6; lane a) were subjected to Western blot analysis by using anti-mammalian eIF6 antibodies as probes. (B) Nuclear and cytoplasmic fractions derived from extracts of KSY607 yeast cells, expressing HA-tagged Tif6p, were preincubated either with calf intestinal alkaline phosphatase (lanes c and d) or with buffer (lanes a and b) and then subjected to Western blot analysis by using anti-HA antibodies as probes. (C) Cell extracts of ³²P-labeled yeast cells expressing either HA-Tif6p (strain KSY607) or untagged Tif6p (strain KSY606) were immunoprecipitated with anti-HA antibodies. The washed immunocomplexes were subjected to SDS-PAGE followed by electrophoretic transfer onto a polyvinylidene difluoride membrane. The blot was first subjected to autoradiography (lanes a and b) and then analyzed by immunoblotting by using anti-HA antibodies (lanes c and d). The faster-migrating band in lane b may represent a degradation product of Tif6p.

FIG. 2.

Purification and characterization of eIF6 kinase from rabbit reticulocyte lysates. (A) A partially purified eIF6 kinase preparation (dialyzed ammonium sulfate fraction derived from the phosphocellulose eluate [P-cell El.]) was subjected to Sephadex G-75 gel filtration as described in Materials and Methods. Aliquots of the protein fractions eluted from the column were assayed for eIF6 kinase activity. (B) The pooled Sephadex G-75 eluate [G75 El.] of eIF6 kinase was subjected to FPLC on a Mono S column. Aliquots of each fraction as well as the pooled G-75 fraction that was loaded onto the Mono S column were assayed for eIF6 kinase activity. Frac. No., fraction number. (C) The pooled Mono S fraction was analyzed by SDS-PAGE followed by silver staining. The major polypeptide band was digested with trypsin and analyzed by mass spectrometry. The sequenced peptides that correspond to CK Iα are indicated. (D) Fractions eluting from the FPLC Mono S column that contained eIF6 kinase activity were analyzed by Western blotting by using anti-mammalian CK Iα antibodies. (E, F, and G) Recombinant human eIF6 (3 pmol) was phosphorylated by using [γ-³²P]ATP and either purified rat liver CK I (E), a purified recombinant mammalian CK Iα isoform (purified rCKIα) (F), or purified recombinant mammalian CK Iδ (purified rCKIδ) (G). In each case, the reaction mixture in lane a did not contain eIF6 while the reaction mixture in lane b was the complete system containing eIF6. In panel E, the slower-migrating bands present in both lanes a and b represent autophosphorylated CK I.

FIG. 3.

Characterization of the phosphorylation sites of mammalian eIF6. (A) Amino acid sequence of mammalian eIF6. The tryptic phosphopeptide that was phosphorylated by purified eIF6 kinase as determined by mass spectrometry is boxed, and Ser-174 and Ser-175 are shown in bold. (B) His-tagged recombinant wild-type (WT) and mutant (S174A; S175A; and S174A, S175A) mammalian eIF6 proteins were purified from IPTG-induced bacterial cell lysates as described in Materials and Methods and then analyzed by SDS-PAGE followed by either Coomassie blue staining or immunoblotting with anti-mammalian eIF6 antibodies. (C) Phosphorylation of recombinant wild-type and mutant (S174A; S175A; and S174A, S175A) mammalian eIF6 proteins by [γ-³²P]ATP and purified rabbit reticulocyte eIF6 kinase (RRL kinase; Mono S fraction). (D) Phosphorylation of recombinant mammalian wild-type eIF6, mutant eIF6 (S174A, S175A), and mutant eIF6 (S166A) by recombinant CK Iδ.

FIG. 4.

Effect of mutation of Tif6p at Ser-174 and Ser-175 on yeast cell growth and viability. (A) Conservation of the amino acid residues surrounding the serine CK I phosphorylation sites of eIF6 (Tif6p). Ser-174 and Ser-175 are in bold. (B) Haploid yeast strain KSY606, carrying an inactive chromosomal TIF6 gene and harboring the wild-type URA3-based TIF6 expression plasmid pRS316-TIF6 that expresses untagged Tif6p, was transformed with different LEU2-based TIF6 expression plasmids. These LEU2 plasmids expressed either HA-tagged wild-type Tif6p (+), HA-tagged mutant Tif6p (Ser-to-Ala or Ser-to-Asp mutation as shown), or no Tif6p ORF (−). Transformants were initially selected on SD−His−Leu−Ura plates (SD−H−L−U) and then replica plated onto (i) SD−His−Leu−Ura and (ii) SD−His−Leu plates with the addition of Ura and 5-FOA (SD−H−L+U+5FOA). The plates were then incubated at 30°C for 3 and 5 days, respectively. (C) Haploid yeast cells harboring both the URA3 plasmid pRS316-TIF6 and the different recombinant LEU2 expression plasmids expressing either the HA-tagged wild-type or HA-tagged mutant (S174A; S175A; and S174A, S175A) Tif6p proteins were recovered from the SD−His−Leu−Ura plates and grown to mid-logarithmic phase in SD medium. Cell lysates were then analyzed by Western blotting by using anti-HA antibodies. Lysates prepared from KSY606 cells that expressed untagged wild-type Tif6p were also analyzed as a control.

FIG. 5.

Effect of mutation of Tif6p at Ser-174 and Ser-175 on its in vivo phosphorylation. The haploid yeast strains described in the legend to Fig. 4C were each grown separately in a 25-ml low-phosphate medium. At A₆₀₀ of 0.5, the cells were treated with 5 mCi of [³²P]orthophosphate (28). Following incubation for 3 h at 30°C, Tif6p was immunoprecipitated (IP) from each cell lysate by using agarose-conjugated anti-HA antibodies. The immunocomplexes formed in each case were washed; subjected to SDS-PAGE, followed by transfer onto a polyvinylidene difluoride membrane; and probed with anti-HA antibodies to detect HA-tagged Tif6p (upper panel). The blot was also analyzed by using autoradiography (lower panel).

FIG. 6.

Subcellular distribution of wild-type Tif6p and the serine phosphorylation mutant of Tif6p. (A, left panel) Lysates of haploid yeast strain UBY608 carrying an inactive chromosomal TIF6 gene and harboring both the TRP1 plasmid pRS314-mycTIF6 (which expresses Myc-tagged Tif6p from its natural promoter) and a recombinant LEU2 plasmid pRS315-TIF6 (S174A, S175A), which expresses mutant HA-Tif6p protein (S174A, S175A) from its natural promoter, were fractionated into postnuclear supernatant (cytosolic fraction) and nuclear pellet as described by Aris and Blobel (1). (Right panel) Similar analysis was carried out with lysates of yeast strain UBY610 expressing both the Myc-tagged Tif6p and theHA-tagged wild-type Tif6p from its natural promoter. Approximately 30 μg of protein from each fraction was subjected to Western blot analysis by using anti-Myc, anti-HA, anti-Nop1, and anti-eIF5 antibodies as probes. (B) Localization of wild-type Myc-Tif6p and wild-type and mutant HA-Tif6p fusion proteins. Indirect immunofluorescence staining was performed with UBY608 cells expressing both the wild-type (WT) Myc-Tif6p (panels a to c) and the mutant HA-Tif6p fusion proteins (panels d to f). Similar indirect immunofluorescence staining was also carried out with UBY610 cells expressing both wild-type Myc-Tif6p and wild-type HA-Tif6p from two different CEN plasmids. Chromatin DNA of yeast cells was stained with DAPI. HA-Tif6p (d and g) was detected by immunofluorescence staining with monoclonal mouse anti-HA antibody and anti-mouse antibodies conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, Oreg.). For Myc-Tif6p (a), the primary antibody used was anti-Myc antibodies and the secondary antibody used was anti-rabbit antibodies conjugated with Alexa Fluor 568 (Molecular Probes). In the third panels (c, f, and i), the two top panels (a and b, d and e, and g and h, respectively) were merged. Arrows indicate the positions of the nuclei.

FIG. 7.

Localization of GFP-tagged wild-type and mutant Tif6p proteins. GFP and DAPI fluorescence of UBY612 cells (expressing GFP-tagged wild-type Tif6p) (a to d) and that of UBY613 cells (expressing GFP-tagged mutant Tif6p) (e to h) are shown. The arrow indicates the position of the cytoplasm.

FIG. 8.

Effect of lack of phosphorylation of Tif6p on its ability to bind to pre-60S ribosomal particles. (A) Cell extract prepared from UBY608 cells expressing both the wild-type Myc-Tif6p and Ala mutant HA-Tif6p was subjected to centrifugation on a 7 to 47% sucrose gradient as described previously (26). Wild-type (WT) Myc-Tif6p (A) and Ala mutant HA-Tif6p (B) in the gradient fractions were detected by immunoblot analysis. (C and D) RNA isolated from each gradient fraction was subjected to Northern blot analysis by using appropriate DNA probes that specifically detect 27S_A+B and 25S rRNAs. The positions of 40S and 60S ribosomal subunits as well as those of 80S ribosomes and polysomes are indicated. Frac. No, fraction number.

FIG. 9.

Effect of mutation of Ser-174 and Ser-175 of Tif6p on its ability to function in pre-rRNA processing. (A) Exponentially growing cultures (100 ml each) of UBY609 {[GAL10::Ub-HA-TIF6] p[LEU2 tif6-GFP(S174A, S175A)]} and strains UBY611 ([GAL10::Ub-HA-TIF6] p[LEU2 TIF6-GFP]) and KSY603 [GAL10::Ub-TIF6] in SGal-Ura medium (A₆₀₀ = 0.5) were transferred to equal volumes of SD-Ura medium and allowed to grow for 120 min. Each culture was centrifuged, suspended in 1 ml of SD-Ura medium, pulsed with 200 μCi of [5,6-³H]uracil for 3 min, and then chased with an excess of unlabeled uracil (1 mg/ml) for the indicated times. A culture of KSY603 grown in SGal-Ura medium was also pulsed and chased in SGal-Ura medium as a control. Total cellular RNA was isolated from each batch of cells. For each time point, an RNA sample containing about 10,000 cpm of ³H radioactivity was analyzed in a 1.2% formaldehyde-agarose gel followed by fluorography as described previously (2). The positions of mature 18S and 25S rRNAs are indicated. (B, left panel) Immunoblot analysis of wild-type GFP-Tif6p and wild-type HA-Tif6p fusion proteins isolated at the indicated times from UBY611 cells following transfer from SGal-Ura medium to SD-Ura medium. (Right panel) Immunoblot analysis of mutant GFP-tagged Tif6p and wild-type HA-tagged Tif6p. Western blot analysis of eIF5 represents loading controls in gels.