Karyopherin escort a variety of macromolecular cargoes between the nucleus and cytoplasm through the nuclear pore. Comprehensive molecular and structural analyses of the NPC, its components, kaps, and other soluble transport factors have yielded tremendous insights into the molecular mechanisms of directional, selective carrier-cargo transport. Importantly, however, our knowledge of which cargoes are transported by each kap remains inadequate. Indeed, nearly one-third of the proteins in the yeast proteome must be translocated into the nucleus at some stage of their life cycle (13
). These include factors involved in regulated gene expression, macromolecular assembly, and nuclear architecture. Understanding regulated transport and the phenotypic consequences of altering transport pathways demands a comprehensive knowledge of the various transport pathways, their cargoes, and their functional redundancy.
Here, we have initiated the large-scale identification of Kap121p cargoes. Overlay blot assays probing an entire nuclear extract identified 27 Kap121p-interacting proteins. Some of these proteins also interacted with Kap123p, supporting the previously reported redundancy between these two kaps (20
). However, 11 proteins preferentially interacted with Kap121p (http://systemsbiology.org/Default.asp?pagename=data
). Given that approximately one-third of all yeast proteins are imported into the nucleus, we believe that this list represents a subset of all Kap121p import cargoes. Indeed, only 2 of the 12 previously characterized Kap121p cargoes were among these 27 proteins (10
). This was not surprising, since the nature of this approach, with a kap as the probe to identify cargoes, demands that the cargoes be sufficiently abundant in order to detect the interactions. In the other cases, Kap121p was identified by using the cargo as the bait (Spo12p) or by monitoring the cargos (Yap1p, Pdr1p, Pho4p, and Aft1p) in different kap mutants. The majority of the Kap-interacting proteins (23 of 27) identified here are abundant factors required for ribosome biogenesis and, as such, represent the most abundant potential Kap121p cargoes.
Amino acid sequence alignments revealed that most of these potential Kap121p import cargoes contained lysine-rich sequences typical of the previously identified NLS sequences of Kap121p cargoes (see reference 30
). Interestingly, although Nop1p contains a weakly related lysine-rich region, it also contains an N-terminal GAR domain that shares significant sequence homology with NLS sequences recognized by another β-kap, Kap104p. Nop1p is an essential nucleolar protein that has remained extremely well conserved, both structurally and functionally (4
), throughout evolution. Nop1p (fibrillarin) is also an autoantigen in humans and has been shown to be implicated in ~8% of the cases of the autoimmune disease scleroderma (5
). Given the unique NLS characteristics of this putative Kap121p transport substrate, the essential functions carried out by Nop1p, and the potential extrapolation to vertebrates, we sought to investigate the Kap121p-Nop1p interaction.
Characterization of the Kap121p-Nop1p interaction demonstrated that Nop1p is a bona fide import cargo for Kap121p. However, Kap121p mediates its import through a direct interaction with the N-terminal rg-NLS. This establishes Nop1p as the first member of a novel class of Kap121p cargoes and extends previous studies implicating the Nop1p GAR domain in nuclear localization (40
). Interestingly, this novel Kap121p-recognized rg-NLS sequence is similar to the rg-NLS sequences of the Kap104p import cargoes Nab2p and Nab4p. This high degree of sequence similarity suggested that these seemingly distinct nuclear transport pathways might converge and mediate the nuclear import of some common cargoes. Genetic studies demonstrated that mutations in both KAP121
are synthetically lethal, establishing a functional relationship between these two β-kaps and strongly suggesting that these two transport pathways overlap. Although rg-NLS sequences were originally characterized for Kap104p, and Kap104p can interact with the rg-NLS of Nop1p in vitro, Kap121p does so with higher affinity and is primarily responsible for its import. Kap104p thus provides an alternative transport route for Nop1p in the absence of Kap121p function.
Why should an NLS be recognized by different kaps? The weaker affinities of each class of NLS for other kaps probably allow kaps to back each other up. This may allow cells to respond to particularly high transport burdens during high growth rates or in response to different environmental conditions, external cues, or developmental stages. Interestingly, recent studies have demonstrated that mutations in KAP121
cause cell cycle defects and that Kap121p transport is specifically attenuated at certain stages of the cell cycle (31
). This has the potential to alter the transport of numerous Kap121p cargoes essential for cell cycle progression. Promiscuity among kap-cargo liaisons creates a situation in which the loss of individual kap function (either temporarily through regulation, or constitutively through genetic perturbation) may only affect a small number of cargoes specific for that kap. Thus, the cell can shut down the transport of classes of molecules by modifying the kap (or a nucleoporin with which it specifically interacts) without affecting all cargoes normally transported by that kap.
Interestingly, the majority of the proteins that interacted specifically with Kap121p in the overlay assay, (Nop1p, Sof1p, Dbp9p, Imp4p, Nop12p, and Rrp12p) are trans
-acting protein factors required for ribosome biogenesis. We followed up on Sof1p because it is an essential nucleolar protein that suppresses a nop1
temperature-sensitive mutant and forms a dimeric complex with Nop1p (21
). We demonstrated that Kap121p mediates the nuclear import of Sof1p through a C-terminal lysine-rich NLS. These studies illustrated that, whereas aa 411 to 450 were sufficient for Kap121p interaction and subsequent import, aa 381 to 489 contained all of the information required for wild-type, nucleolar localization of Sof1p. Furthermore, two nuclear import complexes containing Sof1p could be generated in vitro: Sof1p-Kap121p and Sof1p-Nop1p-Kap121p. Recent studies with mammalian Kap β1 established that this kap mediates the nuclear import of a fragment of kap α and nonclassical NLS-containing cargoes simultaneously (12
), indicating that an individual β-kap can recognize, interact with, and mediate the nuclear import of cargoes containing unrelated NLS sequences. However, in vitro competition assays demonstrated that, unlike mammalian Kap β1, Kap121p does not interact with previously identified lysine-rich Kap121p NLSs and the rg-NLS of Nop1p at the same time. Our data nevertheless indicate that Nop1p and Sof1p can be imported together by Kap121p. In this case, however, Nop1p and Sof1p form a complex that can be recognized by Kap121p, and the trimeric complex traverses the NPC. Solution binding and immunopurification studies showed that under the conditions explored here Kap121p appears to have a higher affinity for the rg-NLS of Nop1p than that present in Sof1p, whereas Sof1p has a higher affinity for Nop1p than Kap121p. Furthermore, expression of NOP1cNLS
chimera or overexpression of KAP104
rescued the mislocalization of Sof1p-GFP in kap121-34
cells, suggesting that Nop1p bridges the interaction between Sof1p and Kap121p.
Recent work from numerous groups has begun to reveal the complex network of interactions between import kaps and their cargoes. The simple idea of one kap for each kind of NLS was soon overturned by several studies showing that, whereas many NLSs are recognized mainly by one kind of kap, they can also be recognized to a lesser extent by other kaps, providing a high degree of redundancy to the nuclear import of many cargoes. As well as further highlighting this kind of redundancy, the present study has added another level of complexity. Here, we show that a single kap (in this case, Kap121p) can recognize both a lysine-rich NLS and an rg-NLS. We had previously assumed that these two very different NLSs would be recognized by different kaps. But it is apparent that cells can exploit surprising flexibility in kap cargo recognition, providing an extra level of redundancy to nuclear transport. Moreover, we also show that associating proteins can be imported as complexes by a single kap. Transporting cargoes in this fashion would ensure that essential, functionally dependent cellular components (such as the rRNA-processing factors transported by Kap121p and Kap104p) are properly localized, in appropriate molar ratios, to the subcellular compartments where they function. Furthermore, we envision that nucleocytoplasmic transport networks like this one would allow the cell to adapt to minor perturbations in individual nuclear transport pathways without drastically affecting other cellular processes. Exploring how these newly discovered levels of transport redundancy are regulated and interconnected is the next challenge in uncovering the network of nuclear transport.