Intersection of the Kap123p-mediated nuclear import and ribosome export pathways - PubMed (original) (raw)

Comparative Study

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways

Y Sydorskyy et al. Mol Cell Biol. 2003 Mar.

Abstract

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

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Figures

FIG. 1.

FIG. 1.

KAP123 interacts genetically with several karyopherins and YGL246C/RAI1. (A) Summary of genetic interactions uncovered by the KAP123 synthetic fitness screen. (B) Top: The chromosomal region contained in the sf17 rescuing plasmid is indicated by the dashed box. Bottom: Plasmids containing candidate genes (RAI1, YGL247W, and PDE1) and genes encoding several karyopherins (SXM1/KAP108, NMD5/KAP119, KAP95, and KAP121) were analyzed using a plasmid shuffling assay in sf17 cells. Note that only cells transformed with YGL246C/RAI1 were able to grow in the absence of pJA8, as assayed by growth on 5-FOA. (C) Top: Cells lacking the RAI1 ORF were viable in a Δ_kap123_ background but exhibited a synthetic fitness interaction with KAP123. Bottom: The reduced growth rate of Δ_rai1_cells was complemented by plasmids encoding either Rai1p or Rai1p-GFP. (D) Sequencing of the RAI1 ORF in the sf17 mutant revealed an A-to-T transversion at position 443, which introduced the nonsense mutation shown.

FIG. 2.

FIG. 2.

Rai1p interacts with Rat1p physically and genetically. (A) Immunoaffinity purification of Rai1-pA from yeast whole-cell lysates yielded stoichiometric amounts of Rat1p. Eluted proteins were resolved by SDS-PAGE and visualized by silver staining. The copurifying band at ∼120 kDa was identified as Rat1p by tandem mass spectromery. (B) Δ_rai1/rat1-1_ cells, relieved of the URA3_-based Rai1p-GFP plasmid by growth on medium containing 5-FOA, were unable to form colonies at all temperatures tested, demonstrating synthetic lethality between these alleles. Shown is the plate incubated at 23°C, with the parental haploid controls Δ_rai1 and rat1-1.

FIG. 3.

FIG. 3.

The localization of Rai1p-GFP to the nucleus is dependent on functional Rat1p. Rai1p-GFP expressed in wild-type cells, detected by fluorescence microscopy in live cells, was predominantly nuclear with a distinct cytoplasmic pool (bottom left). In contrast, when expressed in rat1-1 cells, although grown at permissive temperatures Rai1p-GFP was not concentrated in the nucleus, revealing a predominantly cytoplasmic signal. By comparison, Rat1-GFP remained nuclear or nucleolar when expressed in wild-type or Δ_kap123_ cells. Bar, 5 μm.

FIG. 4.

FIG. 4.

Rai1p interacts with 60S ribosomal subunits. (A) 60S ribosomal subunits were accumulated in the nucleus by expression of a plasmid encoding the nmd3_Δ_100 mutant allele in RAI1-A cells. Under these conditions, Rai1p-pA cofractionated with the 60S ribosomal subunit on sucrose gradients, as determined by immunoblotting the collected fractions to detect the pA tag. Detection of rpL3 served as an internal control to detect 60S particles. The top panel shows the corresponding polysome profile detected by spectrophotometry (OD254) of the fractionated sucrose gradient. (B) Top: Rai1p-pA immunopurifications were performed from wild-type cells and cells expressing nmd3_Δ_100. Proteins bound to Rai1p-pA were eluted with MgCl2 at the concentrations shown. The resulting fractions were immunoblotted to reveal the 60S marker rpL3, which remained associated with Rai1p-pA in cells expressing nmd3_Δ_100. Bottom: Rai1p-pA copurified with a number of 60S ribosomal subunit proteins isolated from cells expressing the nmd3_Δ_100 allele. Proteins contained within the 1 M MgCl2 elution fraction were sequenced by tandem mass spectrometry, leading to the identification of 22 large and 8 small subunit proteins from a total of 33 identifications. Only proteins identified by two or more unique polypeptide matches were considered significant.

FIG. 5.

FIG. 5.

Northern analysis of rRNA processing. Normal rRNA processing is disrupted in strains lacking Rai1p and Kap123p. Equal quantities of total RNA from each strain were separated on a 1% agarose gel or a 10% Tris-borate-EDTA-urea gel in the case of short fragments and hybridized with the oligonucleotide probes listed in Materials and Methods. The position of each probe and the RNA species it is designed to detect are shown at the right.

FIG. 6.

FIG. 6.

Deletion of RAI1 results in a specific 60S subunit assembly defect, while a Δ_kap123/Δ_rai1 double mutant exhibits a decrease in total ribosomal content. Polysome profiles for wild-type (WT) (A), Δ_kap123_ (B), Δ_rai1_ (C), and Δ_kap123/Δ_rai1 (D) cells were obtained by fractionation of cell lysate supernatants on 7-to-42% sucrose gradients and monitoring the OD254. The peaks corresponding to 40S, 60S, 80S, and polysomes are indicated. Because the same amount of sample, as detected by the OD260, was loaded in each case, the peak heights are sensitive indicators of the levels of each subunit. The Δ_rai1_ strain (C) exhibits a deficit of 60S particles as indicated by the reduction in free 60S subunits and the appearance of halfmers. In contrast, the ratio of 40S to 60S particles was normalized in the double mutant Δ_kap123/Δ_rai1; halfmers were reduced, and there was a net decrease in free subunit concentration.

FIG. 7.

FIG. 7.

Cells lacking both Kap123p and Rai1p are defective in 60S subunit export. (A) The faithful integration of GFP reporters into ribosomal particles was confirmed by sucrose gradient centrifugation and subsequent immunoblotting against GFP-tagged reporters and endogenous rpL3/Tcm1p as an internal control. Shown is the profile for rpL2-GFP in an otherwise wild-type background, which is present in 60S, 80S, and polysome fractions, but not the 40S peak. rpL3-GFP and rpL25-GFP profiles were similar (data not shown). (B) Localization of ribosomal-GFP reporters and Nop1-GFP was determined by direct fluorescence microscopy of live cells. Fluorescent signal was detectable throughout the cell in wild-type (WT), Δ_kap123_, and Δ_rai1_ cells but appeared to accumulate in the nucleus and in some cases to the nucleolus in Δ_kap123_/Δ_rai1_ cells. Bar, 5 μm.

FIG. 8.

FIG. 8.

Δ_kap123_/Δ_rai1_ strains accumulate assembled 60S subunits in the nucleus. (A) Nuclear and cytoplasmic fractions were isolated from wild-type (WT) and Δ_kap123_/Δ_rai1_ cells, and 60S components from each subcellular fraction were isolated by further fractionation by sucrose gradient centrifugation under low-Mg2+ conditions. Shown is a histogram of the relative proportion of nuclear and cytoplasmic 60S subunits, quantified by the OD254. Data were normalized to the amount of 60S subunits in the nucleus. (B) Nuclear and cytoplasmic fractions at a 5:1 cellular equivalent load ratio were resolved by SDS-PAGE and probed with monoclonal anti-rpL3 (Tcm1p), as a marker for 60S subunits, and polyclonal anti-Nop1p antibodies, as a control. Results indicate that there is a significant reduction in the levels of rpL3 in the cytoplasm in Δ_kap123_/Δ_rai1_ strains, reflecting the block in 60S subunit export.

FIG. 9.

FIG. 9.

Nmd3p links Kap123p to Rai1p. (A) The sf17 strain (Δ_kap123/rai1-1_) or wild-type (WT) cells were transformed with a plasmid expressing NMD3 or vector alone (pRS425). Growth of these strains on yeast extract-peptone-dextrose (left) or FOA (right) revealed that the increased Nmd3p expression, due to the introduction of NMD3 on the multicopy plasmid, rescues the growth defect associated with sf17 cells (Δ_kap123/rai1-1_). (B) GFP chimeras containing the Nmd3p NLS (amino acids 387 to 435) (26) or the NLS plus an NES (amino acids 387 to 518) were localized in wild-type cells (W303) or W303-derived Δ_kap123_ cells. The Δ_kap123_ strain failed to localize the Nmd3p-NLS-GFP reporter to the nucleus, suggesting that Kap123p is required for efficient import of Nmd3p. Bar, 5 μm.

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