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Originally published In Press as doi:10.1074/mcp.M600309-MCP200 on November 1, 2006.
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Molecular & Cellular Proteomics 6:272-282, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Rapid Evolution Exposes the Boundaries of Domain Structure and Function in Natively Unfolded FG Nucleoporins*,S

Daniel P. Denning{ddagger} and Michael F. Rexach§

From the Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein domains. Here we show that the FG domain of Nups from human, fly, worm, and other yeast species is also enriched in these disorder-associated AAs, indicating that structural disorder is a conserved feature of FG Nups and likely serves an important role in NPC function. Despite the conservation of AA composition, FG Nup sequences from different species show extensive divergence. A comparison of the AA substitution rates of proteins with syntenic orthologs in four Saccharomyces species revealed that FG Nups have evolved at twice the rate of average yeast proteins with most substitutions occurring in sequences between FG repeats. The rapid evolution of FG Nups is poorly explained by parameters known to influence AA substitution rate, such as protein expression level, interactivity, and essentiality; instead their rapid evolution may reflect an intrinsic permissiveness of natively unfolded structures to AA substitutions. The overall lack of AA sequence conservation in FG Nups is sharply contrasted by discrete stretches of conserved sequences. These conserved sequences highlight known karyopherin and nucleoporin binding sites as well as other uncharacterized sites that may have important structural and functional properties.

In eukaryotes, nuclear pore complexes (NPCs)1 regulate the movement of cellular material across the nuclear envelope by functioning as a permeability barrier and as transport machine (1). Small molecules diffuse through NPCs, but large proteins and RNAs (>40 kDa) are excluded unless they contain localization sequences that permit translocation. These targeting signals are recognized by karyopherins (Kaps), mobile receptors that interact with the NPC to facilitate nucleocytoplasmic transport (2). Multiple copies of 30 nucleoporin (Nup) proteins comprise each NPC (3, 4), and approximately half of these Nups are classified as FG Nups due to their content of phenylalanine-glycine (FG) motifs. In Saccharomyces cerevisiae, each FG Nup contains a large domain (150–700 AA in length) composed of FG repeats spaced 10–20 AAs apart. These FG domains function as docking sites for Kaps (5), which bind to phenylalanines in the FG motif (6, 7).

The FG domains of S. cerevisiae Nups are natively unfolded (8, 9). Such "natively unfolded," "intrinsically unstructured," and "disordered" proteins or protein domains lack stable secondary structure and behave as flexible filaments (10, 11). Despite their structural disorder, these domains are often essential for protein-protein and protein-nucleic acid interactions (11). Although the role of disordered structure in FG Nup function is unclear, two hypotheses have been proposed. First the disordered structures in Nups may facilitate rapid translocation of Kap-cargo complexes through the NPC by capturing and releasing Kaps with fast association and dissociation rates. Disordered proteins can exhibit unusually rapid interaction dynamics with binding partners due to a lack of steric limitations (11). In nuclear transport, fast interactions between Kaps and Nups may be necessary for the rapid flux of Kap-cargo complexes through NPCs (12). Thus, the disordered structures of FG Nups might be optimized for highly specific, yet transient interactions with a variety of transport factors. A second hypothesis proposes that the NPC permeability barrier is a meshwork of disordered FG Nup filaments that are interconnected by weak hydrophobic interactions between FG motifs (12). In principle, such a barrier could permit small particles to pass through the interfilament space yet exclude larger molecules from entering the NPC. Large Kap-cargo complexes could gain access by interacting with multiple FG Nups.

If structural disorder in FG Nups serves a critical role in NPC function and architecture, then this feature should be conserved throughout Eukaryotae. In the present study we analyzed the AA composition of FG Nups from evolutionarily distant eukaryotes and found evidence of structural disorder in nearly all FG Nups examined. We also noted that FG Nups have evolved rapidly (particularly in their FG domains) and evaluated the contribution of several parameters to their high amino acid substitution rates. Lastly we identified discrete regions of AA sequence conservation in the FG domains that coincide with known Kap and Nup binding sites and identified clusters of conserved AAs in the regions flanking FG domains that correlate with known NPC anchoring domains and with known molecular interaction sites.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotide and AA Sequences—
The nucleotide and AA sequences of S. cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces bayanus nucleoporins were acquired from the Saccharomyces Genome Database (yeastgenome.org) (13). The AA sequences of human FG Nups (4) were acquired from the Swiss-Prot (us.expasy.ch) and NCBI (ncbi.nlm.nih.gov) databases. The Caenorhabditis elegans FG Nups were identified previously by homology (14). Sequences for C. elegans FG Nups and their corresponding Drosophila melanogaster homologs were acquired from Wormbase (wormbase.org). The sequences of Schizosaccharomyces pombe FG Nups were obtained by searching the S. pombe Gene Data Bank (genedb.org) for FG-containing Nups.

To calculate the fraction of perfectly conserved AAs in yeast FG domains, the FG domains of Nups in S. bayanus, S. mikatae, and S. paradoxus were aligned against the S. cerevisiae FG domain. All of the FG domains used in this study are listed in Supplemental Table S3. The FG domains were defined as the largest contiguous sequence of FG repeats separated by less than 100 AAs and included 10 additional residues flanking the first and last FG motif. Insertions greater than 10 AAs within the FG domains were excluded, such as insertions in the S. paradoxus Nup159 ortholog. Deletions in the FG domains of Nup2 and Nup145 orthologs from S. paradoxus and S. bayanus, respectively, precluded the use of those regions; however, the remaining FG domain sequences available were used. The FG domain of Nsp1 was not included because Nsp1 orthologs were not initially available. FG domains were not defined for Nup53 and Nup59 or their homologs (spomNup40, NPP-19, dmNup35, and hsNup35) because their FG motifs are not clustered into obvious domains.

AA composition, codon adaptation indices (CAIs), and Protein Data Bank homologs for S. cerevisiae proteins were taken from Saccharomyces Genome Database datasets. Protein Data Bank homologs were considered highly significant if a Smith-Waterman sequence analysis yielded a p value <10–100.

Evolution Rate Estimations—
Non-synonymous (dN) and synonymous (dS) substitution rates (the frequency of non-synonymous substitutions per non-synonymous site and the frequency of synonymous substitutions per synonymous site, respectively) for 2,956 proteins were calculated by Wall et al. (15) using gene sequences from S. cerevisiae, S. bayanus, S. mikatae, and S. paradoxus (13). This dataset includes only genes with syntenic orthologs in all four yeast species with >80% coding sequence alignment and excludes ortholog sets with frameshifts caused by insertions or deletions.

AA Sequence Alignments—
The AA sequence alignments of Nups and Kaps from the four Saccharomyces species were generated using the Synteny Viewer program in the "Homology and Comparisons" section of the Saccharomyces Genome Database website (yeastgenome.org). The complete NSP1 sequences were identified after removing an intron and were aligned manually.

Protein Translation Rates, Interactivity, and Dispensability—
Translation rates for 2,700 S. cerevisiae proteins were acquired from Arava et al. (16); the rates were estimated from the number of ribosomes that co-purify with mRNA transcripts. Protein interaction data were obtained from a compilation of large scale protein interaction screens, including yeast two-hybrid screens and biochemical tandem affinity purifications (17). For our analyses, protein-protein interactions were included only if they were identified independently in two or more of the large scale interaction screens.

S. cerevisiae proteins were identified as "essential" or "slow growth" based on results from the systematic deletion of S. cerevisiae genes (18, 19). Yeast lacking essential proteins are inviable on rich medium at 30 °C, whereas yeast lacking slow growth proteins grow at a reduced rate compared with wild-type yeast (19).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The AA Composition of Most FG Nups Is Consistent with Structural Disorder—
We showed previously that the large FG domains of S. cerevisiae Nups are natively unfolded and are enriched in AAs that are common in disordered protein domains (8, 9). Generally disordered polypeptides are enriched in charged and polar AAs (Ala, Arg, Gln, Glu, Gly, Lys, Pro, and Ser) and are depleted of hydrophobic AAs (Asn, Cys, Ile, Leu, Phe, Trp, Tyr, and Val) (11); these two groups of AAs have been referred to as "disorder"-associated and "order"-associated, respectively. To examine whether structural disorder is a conserved feature of FG Nups in other eukaryotes, we determined the disorder- and order-associated AA content of FG Nups from Homo sapiens, D. melanogaster, C. elegans, S. pombe, S. paradoxus, S. mikatae, and S. bayanus. We found that nearly all FG Nups in these organisms exhibit a similar enrichment of disorder-associated AAs (Table I and Supplemental Table S3) as observed previously with the S. cerevisiae FG Nups (9). In particular, the FG domains show a higher concentration of disorder-associated AAs compared with the full-length FG Nup sequences, indicating that the FG domains are more likely to adopt disordered structure than non-FG domains. Thus, it appears that structural disorder of FG Nups is evolutionarily conserved.


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  		TABLE I The percent frequency of disorder- and order-associated AAs of eight proteomes and their known or predicted FG Nups (and their FG domain) and non-FG Nups

 
The Rapid Evolution of FG Nups—
Despite the abundance of FG repeats, similar AA compositions, and highly analogous functions in nuclear transport, the primary structures of FG Nups are poorly conserved over large evolutionary distances, making it difficult to identify orthologous genes in distantly related species (data not shown). In contrast, Kaps are well conserved between species; for example, the yeast Kap95 and human importin-ß share 52% similarity over 99% of their sequence. Thus, it seems that the FG Nups have diverged significantly, whereas their Kap binding partners have not.

The poor sequence conservation between FG Nups throughout Eukaryotae suggests that FG Nups have evolved rapidly. To assess this, we compared the evolution rates of Saccharomyces FG Nups to those of thousands of other Saccharomyces proteins using AA substitution rates (dN) determined by Wall et al. (15). These rates were calculated using sequence alignments of syntenic orthologs from four Saccharomyces species: S. cerevisiae, S. paradoxus, S. mikatae, and S. bayanus. The analysis showed that FG Nups have evolved on average 2 times faster than the mean yeast protein, 2 times faster than non-FG Nups, 3 times faster than Kaps, and 5 times faster than a set of 105 structured proteins (Fig. 1A).


Figure 1
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  		FIG. 1. The FG Nups in budding yeast have evolved rapidly. A, the mean AA substitution rates for FG Nups, non-FG Nups, Kaps, a set of 105 yeast proteins with highly significant homologs in the Protein Data Bank of NMR and crystal structures, and all yeast proteins were derived from alignments of syntenic orthologs from S. cerevisiae, S. paradoxus, S. mikatae, and S. bayanus (analysis of variance, p = 2.2 x 10–7). Error bars represent 95% confidence intervals for each mean. B, the fraction of conserved AAs in the FG domains and non-FG domains calculated from alignments of the FG Nups of four Saccharomyces species.

 
Rapid protein evolution rates can be due either to weak purifying (negative) selection or to positive (adaptive) selection for advantageous amino acid substitutions. To distinguish between these possibilities, we examined the ratio of non-synonymous and synonymous substitution rates (dN/dS) for each yeast FG Nup: a dN/dS ratio <1 is evidence for purifying selection, a ratio =1 indicates neutral evolution, and a ratio >1 is evidence for positive selection. We examined the dN and dS values calculated by Wall et al. (15) for each FG Nup and observed that all of the FG Nups have dN/dS ratios less than or equal to 0.3 with a mean value of 0.2 (data not shown). The small ratios indicate that the FG Nups on average are under purifying selection, not positive selection. However, this analysis assumes a constant dN/dS ratio for the entire length of each FG Nup and might therefore mask individual codons subject to positive selection. A recent study examined 4,133 aligned genes from S. cerevisiae and S. paradoxus to identify genes with codons under positive selection (20). By allowing dN and dS to vary within each gene, this analysis identified 126 proteins, including Nup42 but no other Nups, with at least one codon under positive selection. We conclude that the vast majority of codons within the FG Nups are not under positive selection. Instead their overall rapid evolution rates likely reflect weak constraints on amino acid usage at most sites within their sequences.

A comparison of the non-identical AA sites in alignments of FG Nups from the four Saccharomyces species showed that divergent sites occur more frequently within FG domains than in non-FG domains (Fig. 1B). Indeed the low AA sequence conservation in the FG domains of Nups contrasts with the high level of conservation in Kaps and non-FG Nups (Figs. 1A and 3). Notably AA substitutions were highest among the FG domains of Nups located in peripheral NPC structures (e.g. Nup159, Nup42, Nup60, Nup1, and Nup2, which form the cytoplasmic fibers and nuclear basket) and were lowest among FG domains of Nups located in the central transport conduit (e.g. Nup116, Nup100, Nup57, and Nup49) (Fig. 1B).


Figure 3
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  		FIG. 3. AA sequence alignment of Nups and a Kap from four Saccharomyces species. Identical AAs in all four species are depicted in black bold font, whereas AAs substituted in at least one yeast are depicted in gray. Contiguous conserved sequences of ≥4 identical AA are boxed in yellow and numbered at top left for reference. Yellow boxes are extended in cases where a single non-identical AA separates two or more conserved AAs. Conserved Phe residues are highlighted in red bold font, and non-conserved Phe residues are orange. Clusters of conserved boxes are grouped in blue for non-FG domains and in red for FG domains. Extended FG domains (demarcated by the thin red line boxes) contain the FG domain plus flanking regions that are also highly substituted. Clusters in non-FG domains are separated when ≥10 contiguous, non-conserved AAs lie in between them. Nup53 and Nup59 have no clear FG domain; thus, only clusters were assigned. Dotted blue boxes mark the boundaries of previously characterized domains

 
An alignment of three major types of FG motifs (SAFGXPSFG, GLFG, and FXFG) in Nups from the four Saccharomyces species showed that the majority of the divergent sites occur in the spacer regions that separate the FG motifs (Fig. 2). In these four species, a low percentage (~35%) of amino acids are perfectly conserved in sequences flanking SAFGXPSFG motifs at positions –10 to –4 and +4 to +10 in relation to the phenylalanine (top panel). Similarly ~50 and ~35% of those positions are conserved in sequences flanking the GLFG and FXFG motifs, respectively (middle panels). In contrast, the Phe and Gly residues in FG motifs are under strong purifying selection as ~90% of the phenylalanines and ~80% of the glycines are conserved (bottom panel). This is consistent with crystal structures of the Kap-Nup interaction that show that the phenylalanine in FG motifs is the key binding determinant for Kaps (6). The conserved glycine residue also seems to be important for most FG motifs except for the FXFG motifs in Nup1 and Nup60 that lack or show poor conservation of the glycine residue (data not shown). Last the AAs immediately preceding the FG motifs at positions –3, –2, and –1 are also conserved (all panels). These positions classify FG motifs into subtypes, such as GLFG and FXFG, and may be important in determining the strength and specificity of interactions with Kaps.


Figure 2
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  		FIG. 2. The fraction of AA residues conserved in four Saccharomyces species at sites flanking FG motifs. AA sequences of Nups from four Saccharomyces species were first aligned to identify conserved AA residues flanking each FG motif. The FG motifs from S. cerevisiae FG Nups (and their flanking sequences) were then aligned by subtype (i.e. GLFG, FXFG, etc.) using the phenylalanine(s) in each motif as reference point. The alignments included 10 AAs upstream (–10) and downstream (+10) from the phenylalanine(s) (position 0) in the FG motif. The number (n) of FG motifs used for each alignment is shown for each case.

 
"Islands" of AA Sequence Conservation in the FG Domains of Nups—
The overall lack of AA conservation in the FG domains of Nups is contrasted by the presence of small, discrete stretches of conserved AAs with perfect sequence identity in all four yeast FG Nup orthologs. These islands of conserved AA sequences are present throughout the FG domains and are highlighted in yellow boxes in Fig. 3 and Supplemental Fig. S6. Typically the islands are 6–11 AA in length and center on a single conserved FG motif, although a minority of the islands include conserved AA sequences in between two FG motifs (Supplemental Fig. S7).

The length of most conserved islands in the FG domains (~8 residues long) is similar to the length of Nup1 sequences that contact Kap95 in co-crystals (6, 7, 21), suggesting that each of the conserved islands within the FG domains might function as a contact site for Kaps. Kap95 binds to the C terminus of Nup1 with high affinity (22) by making contact with two stretches of 13 and 6 AAs (AAs 975–987 and 1004–1009) (21). These contact sites are clearly delineated by conserved Boxes 22 and 23 in the otherwise highly substituted FG domain of Nup1 (Fig. 3). Box 22 contains 9 conserved AAs between two conserved Phe residues, and Box 23 has 4 conserved AAs flanking an FG motif. These results suggest that pairs of Phe residues linked by conserved AAs may represent high affinity binding sites for Kaps. Similarly the nine SAFGXPSFG motifs in Nup159 and Nup42 are also highly conserved (Fig. 2); these motifs contain two closely positioned FG repeats and provide high affinity binding sites for the exportin Crm1.2

It is also interesting to note that the centrally located GLFG Nups (Nup49, Nup57, Nup100, Nup116, and nNup145) contain stretches of conserved AAs at their N termini with two or more FG motifs (Supplemental Fig. S7); these stretches might be high affinity binding sites for Kaps. Additional examples of two FG motifs linked by conserved AAs can be found in other FG domains of Nups, most notably in Nup100 and Nup116 (Supplemental Fig. S7). For example, Box 6 in the FG domain of Nup116 (Fig. 3) contains two FG repeats and two additional phenylalanines and binds with high affinity to the non-FG Nup Gle2 (23). Finally the high affinity binding site for Kap121 on Nup53 includes Box 10 (Supplemental Fig. S6), which contains conserved AAs between two phenylalanines (24).

We also noted less common but potentially important FG motifs in the Nups of the four Saccharomyces species (Supplemental Fig. S8). These include the SLFG motif (in Nup100, Nup116, Nup49, nNup145, and Nsp1), the SPFG motif (in Nup42, Nup100, and Nup116), the SFG motif (in Nsp1, Nup49, and Nup1), the NXFG motif (in Nup49, Nup57, Nup116, Nup100, and Nsp1), the WLFG motif (in Nup53 and Nup59), and the FXXFG motif (in Nup159, Nup42, Nup116, Nup100, Nup2, and Nup59). Two additional compound FG motifs, sequences with closely spaced FG repeats, were also noted: the triple FG motif in Nup159 and Nup42 and the quadruple Phe motif in Nup53 and Nup59 (Supplemental Fig. S8). In principle, each FG motif might mediate a specific set of Kap interactions and may specify the strength of these interactions as observed previously for entire FG domains (5). The quadruple Phe motif in Nup53 and Nup59 may be part of a predicted RNA recognition motif (RRM) fold and may function in homodimer formation (25). Other stretches of conserved sequence without FG motifs also exist in the various FG domains (Fig. 3 and Supplemental Fig. S6); these sequences may represent non-canonical binding sites for Kaps or may delineate binding sites for Nups or other nuclear transport factors.

AA Sequence Conservation in Non-FG Domains of Nups—
In comparison with the high AA substitution rates observed for the FG domains of Nups, most of the non-FG domains have retained longer stretches of conserved AAs. The conserved sequence stretches are often contiguous and can be grouped into larger "clusters" of AAs, which are denoted by blue boxes in Fig. 3 and Supplemental Fig. S6. Many of these clusters correspond to domains with previously characterized functional or structural features (Table II and references therein). For example, the conserved Cluster I in Nsp1 and Nup116 clearly defines their respective NPC anchoring domains (26, 27), which are predicted to form coiled coil structures in Nsp1 (27) and a ß-sheet structure in Nup116 (28). Cluster I in the N terminus of Nup159 demarcates a ß-propeller structure that interacts with the DEAD box helicase Dbp5 (29), and Cluster II in Nup2 coincides with its C-terminal Ran binding domain (30). Additional examples of conserved non-FG sequences with characterized functions or structures are shown in Table II, including the binding site for importin-{alpha} (Kap60) at the N terminus of Nup2 (31, 32) and the predicted RRM domains of Nup53 and Nup59 (25). The correlation between conserved AA sequences in FG Nups and domains with previously characterized structure or function implies that other NPC anchoring domains or functionally important domains can be accurately predicted from the alignments shown in Fig. 3 and Supplemental Fig. S6. Based on that premise, we list all characterized and predicted domains of FG Nups in Table II and provide a refinement of their AA boundaries based on the extent of AA conservation.


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  		TABLE II A survey of known structures, functions, and interaction domains for each FG Nup with refined domain boundaries based on AA conservation

Known structures and functions are in regular text; previously predicted structures and functions are in italics; new predictions of structure and function based on AACB value and AA sequence conservation are in bold italics. FG domain plus regions include the FG domain plus flanking, highly substituted sequences. hs RNA, heat shock RNA.

 
The Influence of Protein Expression, Interactivity, and Essentiality on FG Nup Evolution—
To explain the overall fast evolution rate of FG Nups, we examined each for characteristics that correlate with high AA substitution rates, such as low expression level, non-essential function, and low numbers of interacting proteins (33–35). These characteristics contrast with those of highly conserved proteins, which include abundant proteins that must retain codons recognized by abundant tRNAs (36, 37), essential proteins that are required for viability (38, 39), and proteins with many binding partners, which have a high fraction of AA residues under purifying selection (40, 41).

Using available experimental and bioinformatics data, we examined the substitution rate (dN), CAI, translation rate, and protein interactivity (binding partners per protein) of each FG Nup in relation to the S. cerevisiae proteome. As expected, the high percentile rank for FG Nup substitution rates shows that they are among the fastest evolving proteins in the yeast proteome (Fig. 4). In contrast, the FG Nups exhibit a wide range of CAI values, translation rates, and numbers of binding partners. Therefore, none of these parameters appears to explain the high substitution rates of FG Nups, implying that a different property is responsible for their rapid evolution.


Figure 4
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  		FIG. 4. Percentile rank of each FG Nup within the S. cerevisiae proteome for various protein evolution parameters: substitution rate (dN), CAI, translation rate (TR) (proteins/s), and protein interactivity (INT) (binding partners/protein). Each symbol represents the percentile rank of one FG Nup. Data were not available for every FG Nup in some parameters.

 
A Relationship between Structural Disorder and High Evolution Rates—
It is generally assumed that natively unfolded protein domains (such as the FG domains of Nups) are inherently more permissive to AA substitutions than domains with ordered, folded structures. Unfolded protein domains may be subject to less stringent sequence constraints as charged or polar AAs that promote interactions with the aqueous environment may substitute freely without compromising the overall disorder and flexibility of the FG domains. Indeed a study of 26 proteins with disordered domains reported high AA substitution rates in 19 of the 26 (42), suggesting that disordered structures and high AA substitution rates may be generally correlated. To further explore this relationship, we compared the overall structural properties and AA substitution rates of each Nup. To estimate structural content, we adapted the AA classifications determined by Dunker et al. (11) to generate a numerical value that reflects the enrichment of disorder- or order-associated AAs in each Nup. This AA composition bias (AACB) value is expressed as the percentage of disorder-associated AAs in a protein minus the percentage of order-associated AAs. For the S. cerevisiae Nups (FG and non-FG), we observed a strong correlation between AACB value and AA substitution rate (dN) (linear regression: dN = 0.0065(AACB) + 0.168, R2 = 0.369) (Fig. 5A). As expected, Nups with the highest content of order-associated AAs (mostly the non-FG Nups) have fewer AA substitutions than Nups with the highest content of disorder-associated AAs (mostly the FG Nups). In contrast, a set of 105 yeast proteins with folded structures yielded a weak correlation between substitution rate and AACB value (Fig. 5B; linear regression: dN = –0.0014(AACB) + 0.076, R2 = 0.026). In addition, the mean dN value for the set of folded proteins is 0.06 ± 0.01, more than 5 times lower than the mean substitution rate for the FG Nups (Fig. 1A). Finally when the entire S. cerevisiae proteome was binned into quartiles based on AACB value, the 25% of proteins most biased for disorder-associated AAs show a significantly higher substitution rate than proteins near the median AACB value (data not shown). This correlation between disorder-associated AA composition and high substitution rate held true even when the data were controlled for the effects of translation rate, codon usage, and protein dispensability (data not shown). Thus, high substitution rates appear to be a general feature of disordered proteins, and this may explain the unusually rapid evolution rates of the FG Nups.


Figure 5
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  		FIG. 5. The relationship between AA composition and AA substitution rate in Saccharomyces Nups. The AACB value is the percent frequency of disorder-associated AAs (Ala, Arg, Gln, Glu, Gly, Lys, Pro, and Ser) subtracted by the percent frequency of order-associated AAs (Asn, Cys, Ile, Leu, Phe, Trp, Tyr, and Val) in a protein. Proteins with a high content of disorder- or order-associated AAs have positive or negative AACB values, respectively. A, the AA substitution rate (dN) of Saccharomyces Nups (FG and non-FG Nups) correlates with their AACB (linear regression: dN = 0.0065(AACB) + 0.168, R2 = 0.369). Substitution rates were determined as in Fig. 1A. B, Saccharomyces proteins with highly significant homologs in the Protein Data Bank (PDB) of NMR and crystal structures were analyzed as in A (linear regression: dN = –0.00144(AACB) + 0.076, R2 = 0.026). The dashed line indicates the median AACB value for the S. cerevisiae proteome.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Structural Disorder in FG Nups—
Our analyses of the AA compositions of Nups from the yeast, worm, fly, and human proteomes suggest that structural disorder is a conserved feature of the FG Nups in distantly related eukaryotes. Their unusually high content of charged and polar AAs (Table I and Supplemental Table S3) is a hallmark of disordered proteins in general (11). Biophysical experiments will be necessary to test conclusively the structural characteristics of each FG Nup in higher eukaryotes, but recent studies support our prediction that their FG domains also exist in a natively unfolded state (43–45). The apparent conservation of structural disorder in FG domains of Nups throughout Eukaryotae indicates that their inherent structural flexibility plays an important role in NPC function possibly for efficient capture and release of karyopherin-cargo complexes during transport across the NPC or as architectural elements of a size-selective permeability barrier.

The Natively Unfolded FG Domains of Nups Have Evolved Rapidly—
We observed that the AA sequences of Saccharomyces FG Nups exhibit rapid evolution rates compared with the majority of yeast proteins, including non-FG Nups, Kaps, and a set of structured proteins (Figs. 1A, 3, and 5 and Supplemental Fig. S6). The FG domains are highly substituted (Fig. 1B) yet are apparently constrained to retain at least two features: (i) FG motifs (Fig. 2) and (ii) a high density of polar and/or charged AAs surrounding the FG motifs (Table I and Supplemental Table S3). These observations define a basic FG domain prototype that consists of FG motifs separated by 10–20 hydrophilic AAs. Indeed it appears that the intervening sequences between FG motifs are permissive to AA substitutions as long as the physicochemical properties of the region are hydrophilic. Thus, the disordered structures of the FG domains may not require a specific sequence between FG motifs but rather a simple preponderance of hydrophilic AAs that promote extensive solvent interactions and inhibit the formation of stable secondary structure. Only AA sequences that contact interacting proteins, such as the FG motifs that bind to Kaps, are under selective pressure to be conserved (Table II).

To explain the high AA substitution rates of FG Nups, we examined several parameters known to influence protein evolution (Fig. 4). We concluded that neither the protein expression level, nor the interactivity, nor the essentiality of the FG Nups adequately explains their rapid evolution. Instead, a "structural disorder content" parameter showed a significant correlation with protein evolution rate, suggesting that structural disorder permits the high rate of AA substitution in FG Nups and possibly other natively unfolded proteins. High protein evolution rates have been observed for other disordered proteins (42). Importantly this suggests that the structural properties of a protein, in addition to its function, expression level, and contribution to organism viability, can influence its AA substitution rate.

Islands of AA Sequence Conservation in the Natively Unfolded FG Domains of Nups—
Given their lack of stable structure, it is possible that the FG domains display their protein binding determinants as compact, linear sequences like the binding sites for proteins in DNA elements. Many small, discrete islands of AA sequences from 6 to 11 AA in length have been conserved in the FG domains of Nups during yeast evolution (Fig. 3 and Supplemental Fig. S6). These sequences often contain a single FG motif, although some conserved sequences intervene two FG repeats (Supplemental Fig. S7). In principle, each conserved island could represent a binding site for Kaps, Nups, or other proteins involved in NPC function. Within the islands of AA sequence conservation we also identified several types of FG motifs that were previously unrecognized, including the SLFG, SPFG, NXFG, and FXXFG motifs, and a triple FG motif (Supplemental Fig. S8). These FG motifs may interact with different Kaps, of which there are at least 15 in yeast, or may specify different binding affinities for them.

Conserved Domains of Structure and Function in the FG Nups—
We found that all previously characterized structural and functional domains in the S. cerevisiae FG Nups are highly conserved (Table II) despite the overall high AA substitution rates in these Nups. Notable conserved regions include the NPC anchoring domains of Nsp1, Nup42, and Nup116; the ß-propeller structure that binds Dbp5 in the N terminus of Nup159; the Gle2 binding sequence (GLEBS) domain in Nup116; the Ran binding domain in Nup2; the high affinity Kap95 binding site in the C terminus of Nup1; the predicted RRM fold in Nup53 and Nup59; and the high affinity Kap60 binding sites in Nup2 and Nup1 (Table II). This suggests that other conserved, yet uncharacterized domains in FG Nups may have important functions and structures. Based on that premise, our analysis predicts specific AA boundaries for novel structural and/or functional domains in FG Nups and offers refined AA boundaries for known NPC anchoring domains and Kap or Nup binding sites (Table II).

Using AA Sequence Conservation and AA Composition Data to Predict Domain Structure and Function in Natively Unfolded Proteins—
For the yeast FG Nups, there appear to be four different combinations of AA composition and AA substitution rates that produce different structure-function predictions for protein domains. First, a high content of order-associated AAs (AACB value ≤19) and a low AA substitution rate (≥65% AA sequence conservation) predict a folded domain with a conserved molecular interaction; all domains listed as clusters in Table II fall under this category except for Cluster I in Nup60 and Nup53, which have higher AACB values. Second, a high content of order-associated AAs and a high AA substitution rate (≤35% AA conservation) predict a folded domain that may function mainly as a structural bridge or spacer between two domains; the regions between Clusters I and II in Nup59 and between Clusters II and III in Nup159 exemplify this. Third, a high content of disorder-associated amino acids (AACB value >19) and a low AA substitution rate predict a domain that is natively unfolded and participates in molecular interactions; Cluster I in Nup60 and Nup2 and Box12 in Nup60 are clear examples. Fourth, a high content of disorder-associated AAs and a high AA substitution rate predict a natively unfolded domain with molecular interaction sites that are limited to discrete islands of conserved AA sequences; all of the FG domains of Nups and the N termini of Nup1, Nup60, and Nup59 fall under this category. A similar domain, but without discrete islands of conserved AAs, may function as an unstructured flexible linker or spacer between two domains; the AA sequences between clusters in Nup53 and between Cluster I and Box 12 in Nup60 fall under this category.

In summary, we compiled more than 40 experimentally characterized or computationally predicted structural and functional domains in the FG Nups (Table II), and in each case, the local AACB value and the AA conservation value accurately match the local presence or absence of protein structure or function. The overall success of this analysis gave us confidence to make novel predictions regarding the structure and function of uncharacterized domains; our predictions are listed above and are highlighted in bold letters in Table II. Detailed structural, biochemical, and genetic characterizations of these FG Nup domains will be needed to further validate the predictive power of this approach.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Aaron Hirsh for providing the Saccharomyces substitution rate dataset prior to its publication, and we thank Drs. Dmitri Petrov and Arend Sidow for helpful conversations. We also thank Samir Patel, Jessica Wright, and anonymous reviewers for comments on the manuscript.


  FOOTNOTES
 
Received, August 11, 2006, and in revised form, October 13, 2006.

Published, MCP Papers in Press, November 1, 2006, DOI 10.1074/mcp.M600309-MCP200

1 The abbreviations used are: NPC, nuclear pore complex; Nup, nucleoporin; FG Nup, nucleoporin with phenylalanine-glycine repeats; FG domain, FG repeat region of a Nup; Kap, karyopherin; AA, amino acid; RRM, RNA recognition motif; AACB, AA composition bias; CAI, codon adaptation index. Back

2 L. Liu, C. Deng, and M. Rexach, personal communication. Back

* This work was supported in part by National Institutes of Health Grant GM061900 (to M R.) and a National Science Foundation predoctoral fellowship (to D P D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} Present address: Dept. of Biology, Massachusetts Inst. of Technology, Cambridge, MA 02139. Back

§ To whom correspondence should be addressed. Tel.: 831-459-5049; Fax: 831-459-3139; E-mail: rexach{at}biology.ucsc.edu


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rout, M. P., Aitchison, J. D., Magnasco, M., and Chait, B. T. (2003) Virtual gating and nuclear transport: the hole picture. Trends Cell Biol. 13, 622 –628[CrossRef][Medline]

  2. Chook, Y., and Blobel, G. (2001) Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11, 703 –715[CrossRef][Medline]

  3. Rout, M. P., Aitchison, J. D., Suprapto, A., Hjertaas, K., Zhao, Y., and Chait, B. T. (2000) The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635 –651[Abstract/Free Full Text]

  4. Cronshaw, J. M., Krutchinsky, A. N., Zhang, W., Chait, B. T., and Matunis, M. J. (2002) Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915 –927[Abstract/Free Full Text]

  5. Allen, N., Huang, L., Burlingame, A., and Rexach, M. (2001) Proteomic analysis of nucleoporin interacting proteins. J. Biol. Chem. 276, 29268 –29274[Abstract/Free Full Text]

  6. Bayliss, R., Littlewood, T., and Stewart, M. (2000) Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell 102, 99 –108[CrossRef][Medline]

  7. Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R., and Stewart, M. (2002) GLFG and FxFG nucleoporins bind to overlapping sites on importin-ß. J. Biol. Chem. 277, 50597 –50606[Abstract/Free Full Text]

  8. Denning, D. P., Uversky, V., Patel, S. S., Fink, A. L., and Rexach, M. (2002) The S. cerevisiae nucleoporin Nup2p is a natively unfolded protein. J. Biol. Chem. 277, 33447 –33455[Abstract/Free Full Text]

  9. Denning, D. P., Patel, S. S., Uversky, V., Fink, A. L., and Rexach, M. (2003) Disorder in the nuclear pore complex: The FG repeat regions of nucleoporins are natively unfolded. Proc. Natl. Acad. Sci. U S A. 100, 2450 –2455[Abstract/Free Full Text]

  10. Uversky, V. N. (2002) What does it mean to be natively unfolded? Eur. J. Biochem. 269, 1 –10

  11. Dunker, A. K., Lawson, J. D., Brown, C. J., Williams, R. M., Romero, P., Oh, J. S., Oldfield, C. J., Campen, A. M., Ratliff, C. M., Hipps, K. W., Ausio, J., Nissen, M. S., Reeves, R., Kang, C., Kissinger, C. R., Bailey, R. W., Griswold, M. D., Chiu, W., Garner, E. C., and Obradovic, Z. (2001) Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26 –59[CrossRef][Medline]

  12. Ribbeck, K., and Gorlich, D. (2001) Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320 –1330[CrossRef][Medline]

  13. Kellis, M., Patterson, N., Endrizzi, M., Birren, B., and Lander, E. S. (2003) Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423, 241 –254[CrossRef][Medline]

  14. Galy, V., Mattaj, I. W., and Askjaer, P. (2003) Caenorhabditis elegans nucleoporins Nup93 and Nup205 determine the limit of nuclear pore complex size exclusion in vivo. Mol. Biol. Cell 14, 5104 –5115[Abstract/Free Full Text]

  15. Wall, D. P., Hirsh, A. E., Fraser, H. B., Kumm, J., Giaever, G., Eisen, M. B., and Feldman, M. W. (2005) Functional genomic analysis of the rates of protein evolution. Proc. Natl. Acad. Sci. U S A. 102, 5483 –5488[Abstract/Free Full Text]

  16. Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O., and Herschlag, D. (2003) Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U S A. 100, 3889 –3894[Abstract/Free Full Text]

  17. von Mering, C., Krause, R., Snel, B., Cornell, M., Oliver, S. G., Fields, S., and Bork, P. (2002) Comparative assessment of large-scale data sets of protein-protein interactions. Nature 417, 399 –403[Medline]

  18. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901 –906[Abstract/Free Full Text]

  19. Giaever, G., Chu, A. M., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387 –391[CrossRef][Medline]

  20. Yang, Z. (2006) On the varied pattern of evolution of two fungal genomes: a critique of Hughes and Friedman. Mol. Biol. Evol. 23, 2279 –2282[Abstract/Free Full Text]

  21. Liu, S. M., and Stewart, M. (2005) Structural basis for the high affinity binding of nucleoporin Nup1p to the Saccharomyces cerevisiae importin-ß homologue, Kap95p. J. Mol. Biol. 349, 515 –525[CrossRef][Medline]

  22. Pyhtila, B., and Rexach, M. (2003) A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex. J. Biol. Chem. 278, 42699 –42709[Abstract/Free Full Text]

  23. Bailer, S. M., Siniossoglou, S., Podtelejnikov, A., Hellwig, A., Mann, M., and Hurt, E. (1998) Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p. EMBO J. 17, 1107 –1119[CrossRef][Medline]

  24. Lusk, C., Makhnevych, T., Marelli, M., Aitchison, J. D., and Wozniak, R. (2002) Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes. J. Cell Biol. 159, 267 –278[Abstract/Free Full Text]

  25. Handa, N., Kukimoto-Niino, M., Akasaka, R., Kishishita, A., Murayama, K., Terada, T., Inoue, M., Kigawa, T., Kose, S., Imamoto, N., Tanaka, A., Hayashisaki, Y., Shirouzo, M., and Yokoyama, S. (2006) The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain. J. Mol. Biol. 363, 114 –124[CrossRef][Medline]

  26. Bailer, S. M., Balduf, C., Katahira, J., Podtelejnikov, A., Rollenhagen, C., Mann, M., Pante, N., and Hurt, E. (2000) Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex. J. Biol. Chem. 275, 23540 –23549[Abstract/Free Full Text]

  27. Bailer, S. M., Balduf, C., and Hurt, E. (2001) The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport. Mol. Cell Biol. 21, 7944 –7955[Abstract/Free Full Text]

  28. Robinson, M., Park, S., Sun, Z., Silver, P., and Wagner, G. (2005) Multiple conformations in the ligand-binding site of the yeast nuclear pore-targeting domain of Nup116. J. Biol. Chem. 280, 35723 –35732[Abstract/Free Full Text]

  29. Weirich, C., Erzberger, J., Berger, J., and Weis, K. (2006) The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore. Mol. Cell 16, 749 –760

  30. Dingwall, C., Kandels-Lewis, S., and Seraphin, B. (1995) A family of Ran binding proteins that includes nucleoporins. Proc. Natl. Acad. Sci. U S A. 92, 7525 –7529[Abstract/Free Full Text]

  31. Gilchrist, D., and Rexach, M. (2003) Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm. J. Biol. Chem. 278, 51937 –51949[Abstract/Free Full Text]

  32. Matsuura, Y., Lange, A., Harreman, M. T., Corbett, A. H., and Stewart, M. (2003) Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import. EMBO J. 22, 5358 –5369[CrossRef][Medline]

  33. Pal, C., Papp, B., and Hurst, L. D. (2003) Rate of evolution and gene dispensability. Nature 421, 496 –497[Medline]

  34. Pal, C., Papp, B., and Hurst, L. D. (2001) Highly expressed genes in yeast evolve slowly. Genetics 158, 927 –931[Free Full Text]

  35. Rocha, E. P. C., and Danchin, A. (2004) An analysis of determinants of amino acid substitution rates in bacterial proteins. Mol. Biol. Evol. 21, 108 –116[Abstract/Free Full Text]

  36. Dong, H., Nilsson, L., and Kurland, C. G. (1996) Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol. 260, 649 –663[CrossRef][Medline]

  37. Futcher, B., Latter, G. I., Mondardo, P., McLaughlin, C. S., and Garrels, J. I. (1999) A sampling of the yeast proteome. Mol. Cell. Biol. 19, 7357 –7368[Abstract/Free Full Text]

  38. Hirsh, A. E., and Fraser, H. B. (2001) Protein dispensability and rate of evolution. Nature 411, 1046 –1049[CrossRef][Medline]

  39. Jordan, I. K., Rogozin, I. B., Wolf, Y. I., and Koonin, E. V. (2002) Essential genes are more evolutionary conserved than are nonessential genes in bacteria. Genome Res. 12, 962 –968[Abstract/Free Full Text]

  40. Fraser, H. B., Hirsh, A. E., Steinmetz, L. M., Scharfe, C., and Feldman, M. W. (2002) Evolutionary rate in the protein interaction network. Science 296, 750 –752[Abstract/Free Full Text]

  41. Fraser, H. B., Wall, D. P., and Hirsh, A. E. (2003) A simple dependence between protein evolution rate and the number of protein-protein interactions. BMC Evol. Biol. 3, 11 –16[CrossRef][Medline]

  42. Brown, C. J., Takayama, S., Campen, A. M., Vise, P., Marshall, T. W., Oldfield, C. J., Williams, C. J., and Dunker, A. K. (2002) Evolutionary rate heterogeneity in proteins with long disordered regions. J. Mol. Evol. 55, 104 –110[CrossRef][Medline]

  43. Buss, F., Kent, H., Stewart, M., Bailer, S. M., and Hanover, J. A. (1994) Role of different domains in the self-association of rat nucleoporin p62. J. Cell Sci. 107, 631 –638[Abstract]

  44. Lim, R. Y., Aebi, U., and Stoffler, D. (2006) From the trap to the basket: getting to the bottom of the nuclear pore complex. Chromosoma 115, 15 –26[CrossRef][Medline]

  45. Lim, R. Y., Huang, N. P., Koser, J., Deng, J., Lau, K. H., Schwarz-Herion, K., Fahrenkrog, B., and Aebi, U. (2006) Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc. Natl. Acad. Sci. U S A. 103, 9512 –9517[Abstract/Free Full Text]

  46. Allen, N. P., Patel, S. S., Huang, L., Chalkley, R. J., Burlingame, A., Lutzmann, M., Hurt, E., and Rexach, M. (2002) Deciphering networks of protein interactions at the nuclear pore complex. Mol. Cell. Proteomics 1, 930 –946[Abstract/Free Full Text]

  47. Gorsch, L. C., Dockendorff, T. C., and Cole, C. N. (1995) A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes. J. Cell Biol. 129, 939 –955[Abstract/Free Full Text]

  48. Del Priore, V., Heath, C., Snay, C., MacMillan, A., Gorsch, L., Dagher, S., and Cole, C. (1997) A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae. J. Cell Sci. 110, 2987 –2999[Abstract]

  49. Devos, D., Dokudovska, S., Williams, R., Alber, F., Eswar, N., Chait, B., Rout, M., and Sali, A. (2006) Simple fold composition and modular architecture of the nuclear pore complex. Proc. Natl. Acad. Sci. U S A. 103, 2172 –2177[Abstract/Free Full Text]

  50. Hurwitz, M. E., Strambio-de-Castillia, C., and Blobel, G. (1998) Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex. Proc. Natl. Acad. Sci. U S A. 95, 11241 –11245[Abstract/Free Full Text]

  51. Strahm, Y., Fahrenkrog, B., Zenklusen, D., Rychner, E., Kantor, J., Rosbach, M., and Stutz, F. (1999) The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr 255p. EMBO J. 18, 5761 –5777[CrossRef][Medline]

  52. Stutz, F., Kantor, J., Zhang, D., McCarthy, T., Neville, M., and Rosbash, M. (1997) The yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its FG-repeat region. Genes Dev. 11, 2857 –2868[Abstract/Free Full Text]

  53. Aitchison, J. D., Rout, M. P., Marelli, M., Blobel, G., and Wozniak, R. W. (1995) Two novel related yeast nucleoporins Nup170p and Nup157p: complementation with the vertebrate homologue Nup155p and functional interactions with the yeast nuclear pore-membrane protein Pom152p. J. Cell Biol. 131, 1133 –1148[Abstract/Free Full Text]

  54. Teixeira, M. T., Fabre, E., and Dujon, B. (1999) Self-catalyzed cleavage of the yeast nucleoporin Nup145p precursor. J. Biol. Chem. 274, 32439 –32444[Abstract/Free Full Text]

  55. Huang, L., Baldwin, M. A., Maltby, D. A., Medzihradszky, K. F., Baker, P. R., Allen, N., Rexach, M., Edmondson, R. D., Campbell, J., Juhasz, P., Martin, S. A., Vestal, M. L., and Burlingame, A. L. (2002) The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry. Mol. Cell. Proteomics 1, 434 –450[Abstract/Free Full Text]

  56. Marelli, M., Lusk, C., Chan, H., Aitchison, J. D., and Wozniak, R. (2001) A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope. J. Cell Biol. 153, 709 –724[Abstract/Free Full Text]

  57. Denning, D., Mykytka, B., Allen, N., Huang, L., Burlingame, A., and Rexach, M. (2001) The nucleoporin Nup60p functions as a Gsp1p-GTP sensitive tether for Nup2p at the nuclear pore complex. J. Cell Bio. 154, 937 –950[Abstract/Free Full Text]

  58. Rexach, M., and Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683 –692[CrossRef][Medline]

  59. Bogerd, A. M., Hoffman, J. A., Amberg, D. C., Fink, G. R., and Davis, L. I. (1994) nup1 mutants exhibit pleiotropic defects in nuclear pore complex function. J. Cell Biol. 127, 319 –332[Abstract/Free Full Text]

  60. Floer, M., Blobel, G., and Rexach, M. (1997) Disassembly of RanGTP-karyopherin ß complex, an intermediate in nuclear protein import. J. Biol. Chem. 272, 19538 –19546[Abstract/Free Full Text]


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