Evolutionary specialization of the nuclear targeting apparatus

Abstract

The alpha- and beta-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All alpha-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various alpha-Kaps fall into two groups, alpha1 and alpha2. Whereas animals encode both types, the yeast genome encodes only an alpha1-Kap. The beta-Kaps are characterized by 14-15 tandemly arranged HEAT motifs. We show that the Arm repeats of alpha-Kaps and the HEAT motifs of beta-Kaps are similar, suggesting that the alpha-Kaps and beta-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of beta-Kaps, consistent with their proposed cargo specificities. We present a model in which an alpha-independent beta-Kap progenitor gave rise to the alpha-dependent beta-Kaps and the alpha-Kaps.

Figure 1

α-Kap alignment. Complete ORFs from the various α-Kaps were aligned by using the clustalv package of programs (27) and high gap penalties. ∗ and • below the alignment indicate identities and similarities, respectively among all α-Kaps. The boxes indicate the most recent definition of the Arm repeats (12, 13), and the horizontal arrows indicate the HEAT motifs. The PID (protein identification) numbers of each sequence are indicated at the beginning of the alignment. Note that the Caenorhabditis elegans α-Kap, a composite of several expressed sequence tags, has a considerably longer N terminus, which may be an artifact.

Figure 2

Conservation of the order of Arm repeats within the α-Kaps. The eight individual Arm repeats from yeast α1-Kap, human α1-Kap, and human α2B-Kap were aligned, and a phylogenetic relationship was derived by using the Neighbor-Joining method (28). The tree shown here is a 50% consensus tree with bootstraps represented as a percentage of 1,000 replications.

Figure 3

Phylogeny of the α-Kaps. The alignment of the Arm repeat-containing domains of the various α-Kaps was used to construct a phylogeny by the Neighbor-Joining method (28). Shown here is the consensus tree of 1,000 bootstrap replications. The tree was midpoint-rooted between the α1 and α2-Kaps. The phylogenetic relationship of the proteins based on the Maximum Parsimony algorithm, by using the paup package of programs (30), also was determined and gave the identical topology. Nomenclatures based both on the submitted names as well as a proposed scheme are indicated.

Figure 4

Distribution of HEAT motifs in the β-Kaps. HEAT motifs are indicated as boxes, and non-HEAT-containing sequences are represented by thick lines. The entire human and yeast ORFs were aligned by clustalv (27) in pairs. A window of about 40 amino acids, matching the HEAT consensus (11), then was used to match the ORF pairs with a high gap penalty, to score for individual HEAT motifs. The alignment windows were evaluated individually on the basis of matches to the residues identified previously as being important for the secondary structure. Mismatches of one important residue per α-helix were allowed as long as the secondary structure prediction was unaffected. Secondary structure predictions were used based on the sspred method (29) to confirm the primary sequence alignments. The darkly shaded boxes represent HEAT motifs that are unambiguously homologous in the various β-Kaps (data not shown). The dashed boxes represent HEAT motifs that are not close matches to the universal HEAT consensus (Fig. 3) but are predicted to assume a similar secondary structure (11, 19, 20). Each HEAT repeat is bounded by numbers above and below the box that indicate the beginning and ending amino acid for that particular repeat. Accession numbers/protein identification numbers are indicated below the protein names.

Figure 5

Alignment of conserved sixth and seventh HEAT motifs of β-Kap sequences. ∗ and • at the bottom of the alignment represent identities and similarities among the β-Kap sequences, respectively. Putative Ran-binding determinants (20) are highlighted in bold.

Figure 6

Phylogeny of the β-Kaps. Neighbor-Joining trees (28) based on the alignment in Fig. 5, with bootstrap values of 1,000 replications. The phylogenetic relationship of the proteins based on Maximum Parsimony (30) differs in that the human Kap121 falls outside a branch containing both the yeast Kap121 and yeast Kap123.

Figure 7

Alignment of Arm and HEAT motifs. HEAT motifs from the various β-Kaps and Arm repeat-containing segments from Yeast α2-Kap and Drosophila Armadillo (β-catenin) were aligned to the universal HEAT consensus (11). p and h indicate polar and hydrophobic residues, and HHH indicates α-helical secondary structure prediction. The arrow indicates the Arm repeat junction identified in Fig. 1 (12). Numbers at the beginning and end of each amino acid stretch represent the number of amino acids to the end of the ORF. Numbers in parentheses represent the number of amino acids between the consecutive HEAT repeats.