Fig. 2. Multiple alignment of selected NTF2-like domains. The sequences are grouped into four subfamilies according to the domain organization of entire proteins (Figure 1): (I) TAP; (II) G3BP; (III) plant protein kinases; and (IV) NTF2. First column, protein names; second column, species names: At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Nc, Neurospora crassa; Nt, Nicotiana tabacum; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Xl, Xenopus laevis; third column, positions of the first aligned residues in each of the sequences; last column, DDBJ/EMBL/GenBank database accession Nos. The positions conserved in 80% of the sequences are indicated in the consensus line: c (DEHKR), charged; h (ACFGHIKLMRTVWY), hydrophobic; l (ILV), aliphatic; p (CDEHKNQRST), polar; s (ACDGNPSTV), small; t (ACDEGHKNQRST), turn like; u (AGS), tiny. The statistically significant alignment blocks verified by MACAW are indicated by the green underlines in the consensus line. The positions conserved among the four subfamilies are indicated in bold characters with colors: cyan, hydrophobic; green, tiny; orange, hydroxyl; black, polar. The random mutations of tripeptides to alanines are highlighted. The designed point mutations that are mentioned in the text are indicated by blue underlines; the colors of the corresponding asterisks indicate the phenotypic effect: red, impaired binding; cyan, no obvious effect. The known secondary structure of rat NTF2 is indicated below the consensus line (H, α-helix; E, β-strand) and agrees well with the predicted secondary structure (Rost and Sander, 1994) of TAP sequences. The surface accessibility is shown below the predicted secondary structure. The dimer contact is shown at the bottom.

Fig. 4. GST pull-down assays with TAP and p15 mutants. (A) Binding assays were performed with [³⁵S]methionine-labeled TAP or various TAP mutants and immobilized GST–p15 on glutathione agarose beads. One tenth of the input (i) and one quarter of the bound fractions (s) were analyzed on SDS–PAGE followed by fluorography. Lanes (b) show the background obtained with glutathione agarose beads coated with GST. (B) Binding assays were performed with [³⁵S]methionine-labeled p15 or various p15 mutants and immobilized GST–TAP on glutathione agarose beads. Symbols are as in (A).

Fig. 1. Modular architecture of proteins with NTF2-like domains. The numbers indicate the lengths of the sequences in residues; the protein names correspond to Figure 2; DDBJ/EMBL/GenBank accession Nos are given in parentheses. The binding partners of TAP and the regions involved in binding defined in previous studies (Braun et al., 1999; Bachi et al., 2000) are indicated under the TAP sequence. Abbreviations: LR, leucine-rich repeat; UBA, ubiquitin-associated domain; NPC, nuclear pore complex; RRM, RNA recognition motif; S-TKc, catalytic domain of serine/threonine protein kinase.

Fig. 3. Proposed model of the interaction between the middle domain of TAP and p15. The model structure was calculated (Sali and Blundell, 1993) based on the 3D structure of the homodimer of NTF2 (Bullock et al., 1996). Cyan, TAP; green, p15. The side chains are shown for positions where mutations were introduced: red, affected binding; dark blue, binding efficiency similar to the wild-type proteins. Residue numbers are indicated for mutation set (ii) (see the text). Numbers below and above 400 denote residues in p15 and TAP, respectively. The triple alanine mutations are shown in gray. The figure was prepared using MOLMOL (Koradi et al., 1996).

Fig. 5. The NPC-binding domain of TAP contains a UBA-like motif. (A) Multiple alignment of UBA domains. Description of the columns is as in Figure 2. Species names (other than those in Figure 2): Mm, Mus musculus; Tt, Thermus thermophilus; Ec, Escherichia coli. Conserved hydrophobic positions are colored in cyan with highly conserved residues in bold characters. Highly conserved positions occupied with negatively charged residues are indicated by bold characters. The mutation, D595R, which interrupts binding to nucleoporins, is indicated by a red asterisk. The alignment consists of two groups: top, UBA domains found in TAP and the sequences with significant similarity; bottom, structurally related elongation factor Ts (EF-Ts). See the legend to Figure 2 about the consensus line. The known secondary structures of 1uba and 1efu are indicated below the consensus line (H, α-helix). The predicted secondary structure, which follows the known secondary structures, was calculated by the PHD program (h and H, α-helix predicted with expected average accuracy below and above 82%, respectively; Rost and Sander, 1994). The surface accessibility below (0 low, 9 high) was calculated using the DSSP program (Kabsch and Sander, 1983). (B) Proposed model for the UBA-like domain of TAP based on the structure of the UBA domain (PDB: 1uba) (Dieckmann et al., 1998. The red side chain indicates the position corresponding to TAP D595. The direction of the chain is indicated at both termini. The figure was prepared using MOLMOL (Koradi et al., 1996). (C) Mutations in the conserved loop of the UBA-like domain disrupt TAP–nucleoporin interaction. The assays were performed using the [³⁵S]methionine-labeled nucleoporins indicated on the left, and recombinant GST, GST–TAP, GST–TAP D595R or GST–TAPΔ567–613 as indicated above the lanes. [³⁵S]methionine-labeled p15 served as an internal control, as its interaction with TAP is not affected by mutations or deletions that prevent nucleoporin binding Bachi et al., 2000). One tenth of the input and one quarter of the bound fractions were analyzed on SDS–PAGE followed by fluorography.