Here, we used a synthetic fitness genetic screen to identify components that support Kap123p function and, when mutated, render cells dependent on the presence of Kap123p. Not surprisingly, this screen yielded several karyopherins: Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. Indeed, Kap121p has previously been shown to compensate for the loss of Kap123p, likely by importing several ribosomal proteins in its absence (46
). In addition, Sxm1p/Kap108p has previously been shown to interact physically with the ribosomal proteins rpL11A/B, rpL25, and rpL31A/B (42
), and overproduction of this karyopherin suppresses a kap121ts
mutant phenotype (41
), suggesting further complementarity between Kap123p, Kap121p, and Sxm1p/Kap108p. Moreover, the high degree of similarity between Sxm1p/Kap108p and Nmd5p/Kap119p (4
) and the identification of NMD5
as genetically interacting with KAP123
suggest a similar and likely overlapping function for this karyopherin. Interestingly, the use of several karyopherins to import different ribosomal proteins by yeast cells seems to be conserved in metazoans. Numerous vertebrate karyopherins, including Kap β/importin β, Kap β2/transportin, RanBP5, RanBP7, and importin 11, have been implicated in ribosomal protein import (22
). Furthermore, a metazoan Kap123p orthologue, importin β4, has recently been characterized (29
). Thus, data from different systems suggest that ribosomal protein import and assembly makes use of a variety of import (and export) karyopherins and that their functional overlap may permit the loss of individual factors. However, it remains unclear to what extent these karyopherins functionally overlap under normal conditions and whether this multiple redundancy is exploited by cells to globally control the transport of classes of different molecules.
Beyond redundant import pathways, the genetic screen revealed an interaction between the ribosome assembly factor RAI1 and KAP123. In an attempt to understand the molecular bases for this interaction, we investigated how the loss of both proteins specifically affected the process of ribosome biogenesis. As shown previously, cells lacking Rai1p revealed a 60S assembly defect, accumulating 27S rRNA and halfmers in polysome profiles. Remarkably, the loss of Kap123p had little effect on ribosome assembly or export, as we detected no obvious rRNA accumulation or ribosomal subunit export defects in cells lacking Kap123p, but cells lacking both Rai1p and Kap123p displayed a more complex phenotype. These cells showed a moderate augmentation of the 27S rRNA defect, normalization of the 40S/60S ratio, an overall decrease in the number of ribosomes, and an accumulation of assembled pre-60S subunits in the nucleus. Furthermore, although other karyopherins import ribosomal proteins, the genetic interaction between Rai1p and Kap123p was specific: out of the five yeast Kaps tested, including all those known to import ribosomal proteins, only KAP123 was able to rescue sf17 cells and only kap123/rai1 mutants displayed pre-60S ribosomal subunit export defects. Together these data suggest that, in these cells, the 60S biogenesis program was attenuated at a late, postassembly, preexport step.
It is evident that cells lacking Rai1p are defective in 60S assembly, but why should the additional loss of Kap123p, whose only known function is in nuclear import, specifically cause an export defect? Consider ribosome assembly as a simple series of chemical reactions; the removal of products at each step contributes to the progression of the entire pathway. Alternatively, the failure to remove products at any step causes the accumulation of intermediates. Thus, considering that Rai1p interacts physically with Rat1p, a late-acting exonuclease, and that Rai1p was detected associated with assembled pre-60S subunits, we hypothesized that the protein(s) required at the late stages of biogenesis is not imported efficiently in kap123
strains and that, in combination with a mutation in RAI1
, products downstream of Rai1p function were not efficiently processed to the next step, leading to the accumulation of (partially) assembled subunits. Thus, we speculate that the specific genetic interaction observed between KAP123
is due to a reduced efficiency of 60S subunit assembly, contributed by a lack Rai1p function, as well as an inability to import critical ribosomal assembly and export factors. Here we show that one such factor is Nmd3p. Overexpression of NMD3
rescued the slow-growth phenotype observed in sf17 (rai1-1/
, and Δrai1
cells (data not shown), and direct visualization of an Nmd3-GFP chimera demonstrated that efficient Nmd3p import into the nucleus requires Kap123p. While the mislocalization of Nmd3p is evident in kap123
cells, deletion of NMD3
is lethal; thus, it is likely that other factors can also import Nmd3p in the absence of Kap123p. Furthermore, it is also likely that inefficient import of other factors contributes to the genetic interaction observed here. One such candidate is rpL10, also a late-acting assembly and export factor for 60S subunits imported by Kap123p (20
). Nevertheless, because NMD3
, but not RPL10
, expression is sufficient to suppress the growth defects associated with rai1/kap123
cells, it is apparent that Nmd3p mislocalization is a primary cause of the rai1/kap123
Surprisingly, among the mutants assayed, only rat1-1
cells mislocalized Rai1p-GFP from the nucleus to the cytoplasm. Considering the tight in vitro binding between Rat1p and Rai1p, we propose that the steady-state localization of Rai1p to the nucleus is a result of its interaction with Rat1p. Furthermore, it is interesting to speculate that Rai1p may be used by Rat1p to tether the assembling subunit, but that, in the absence of a functional Rat1p, Rai1p may exit the nucleus with the ribosomal subunit. It has previously been shown that loss of active Rat1p can be complemented by directing Xrn1p, a normally cytoplasmic exonuclease, to the nucleus (30
). It is not yet known if quality control mechanisms exist to prevent incompletely assembled ribosomal subunits from exiting the nucleus, but it seems possible that under conditions where Rai1p becomes cytoplasmic the subsequent maturation of unprocessed rRNA could occur in the cytoplasm, under the direction of Xrn1p. It will be interesting to determine if the function of Xrn1p is also augmented by Rai1p and if the export defect observed here also results from an active quality control mechanism.
The synthetic fitness screen employed here revealed a complex genetic interaction between KAP123, a nuclear import factor, and RAI1, a ribosome biogenesis factor, which manifests itself in a ribosome subunit export defect. The data support a model where a cause of the defect is an inability to import sufficient quantities of the essential export factor Nmd3p to overcome the loss of Rai1p. It is particularly intriguing to speculate that the coordination of the late steps of 60S biogenesis and nuclear export involve a direct link between Rai1p and Nmd3p, perhaps during the loading of ribosomes with Nmd3p. However, this remains to be investigated. The findings presented here underscore the integration of ribosome assembly and nucleo-cytoplasmic exchange; however, a good understanding of the entanglement between these two pathways demands further identification and characterization of ribosome assembly factors and an understanding of their relationships with the nuclear import-export apparatus.