A sequence motif conserved in diverse nuclear proteins identifies a protein interaction domain utilised for nuclear targeting by human TFIIS

Abstract

The three structural domains of transcription elongation factor TFIIS are conserved from yeast to human. Although the N-terminal domain is not needed for transcriptional activity, a similar sequence has been identified previously in other transcription factors. We found this conserved sequence, the LW motif, in another three human proteins that are predominantly nuclear localized. We investigated two examples to determine whether the LW motif is actually a dedicated nuclear targeting signal. However, in one of the newly identified proteins, hIWS1 (human Iws1), a region containing classic nuclear localization signals (NLS) rather than the LW motif was necessary and sufficient for nuclear targeting in HeLa cells. In contrast, human TFIIS does not possess an NLS and only constructs containing the LW motif were efficiently targeted to nuclei. Moreover, mutations in the motif could cause cytoplasmic accumulation of TFIIS and enabled a structure/function assay for the domain based on the efficiency of nuclear targeting. Finally, GST pull-down assays showed that the LW motif is part of a protein-binding domain. We suggest that the targeting role the LW motif plays in TFIIS arises from its more general function as a protein interaction domain, enabling TFIIS to bind a carrier protein(s) that accomplishes nuclear import.

Figure 1

The LW sequence motif. (A) Identification of the LW motif in diverse human polypeptides. The extent of the LW motif is indicated by arrows above a CLUSTALW 1.7 (47) alignment that was manually adjusted to position any gaps to the interhelical linker regions of the structure predicted for this region of human TFIIS (17). Identical or functionally similar residues are outlined in black and the asterisks at the bottom indicate the invariant residues that we used to define the motif. Amino acid co-ordinates are given at the start of each sequence. Conceptual translation products were predicted from sequences with the following nucleotide database accession numbers: TFIIS isoforms, M81601, D50495, AK027024; elongin A isoforms, L47345, AB030834, AB076840; Med26, AF104253; PPP1R10p, Y13247; PIBP, AK056674; hIWS1, AK027561. (B) Sequence conservation of LW motifs between yeast and human in TFIIS and Iws1. Again, identical or functionally similar residues are outlined in black and the asterisks indicate the invariant residues used to define the motif. Above are indicated the positions of the four helices comprising yeast TFIIS domain I as predicted by Booth et al. (17).

Figure 2

Nuclear targeting and subnuclear localization of hIWS1 and PIBP in HeLa cells. (A) The top panel shows a fluorescent image (and corresponding DIC image) of cells expressing myc-tagged hIWS1 that have been immunostained with mAb 9E10 against the tag. For comparison the central panels show the similar, predominantly nuclear localization pattern exhibited by the myc-tagged human TFIIS.o isoform. The bottom panels are from cells expressing FLAG-tagged PIBP, which again show a predominantly nuclear distribution of the tagged protein. (B) Cells expressing FLAG-tagged PIBP were co-stained for endogenous TFIIS. Apart from a general nuclear staining, the subnuclear localization patterns of the two proteins appear distinctive, with PIBP being prevalent in nucleoli (arrows in DIC image) whereas TFIIS is relatively underrepresented in these organelles.

Figure 3

A region containing NLS sequences but not the LW motif suffices for nuclear targeting of hIWS1 (A) Immunolocalization patterns of myc-tagged hIWS1 region 1–573 (which does not contain the LW motif) compared with regions 573–819 and 573–776 (which do contain the LW motif). Full-length hIWS1 (1–819) is shown as a control. The diagram below indicates the extent of the tagged polypeptides (numbered according to amino acid residues) and the basic organization of hIWS1. The locations of the three potential NLS signals predicted in the hIWS1 sequence are indicated by downward arrows and the location of the LW motif is shown by horizontal arrows. The region containing the highly conserved sequence present in both yeast and human and that includes the LW motif is shaded, and two other well-conserved regions that flank an extensive region possessing many acidic residues are indicated by hatching. (B) Immunolocalization of myc-tagged fusions between regions of hIWS1 and the large cytoplasmic enzyme pyruvate kinase (PK). Immunolocalization of a full-length hIWS1–PK fusion and of PK alone are shown as controls. Above the fluorescent images the hIWS1 fragments used in the fusions are drawn with respect to the overall organization of the polypeptide.

Figure 4

Human TFIIS polypeptides that contain the LW motif show efficient nuclear targeting. The diagram indicates the general organisation of TFIIS and the regions of the hTFIIS.o isoform used in our myc-tagged constructs. The C-terminal region 172–301 is highly conserved, and in terms of the structure/function properties of TFIIS [reviewed in (11)] includes the intact Zn ribbon domain, and a portion of domain II. Region 1–77 contains the highly conserved LW motif and represents that part of domain I with a defined structure. Residues 78–301 can confer all known transcriptional functions of TFIIS. The central panel shows immunofluorescent images of cells expressing the corresponding myc-tagged constructs stained with mAb 9E10. The lower panel shows immunoblots of deletion products and full-length TFIIS from transfected HeLa cell extracts probed with mAb 9E10. un = control extract from untransfected cells.

Figure 5

Effect of LW motif mutations on the nuclear targeting efficiency of TFIIS. The sequence of hTFIIS.o is shown with conserved and invariant residues indicated as in Figure 1A and with the locations of nine substitution mutations (black dots) and a deletion mutation (hNΔ30; broken line) also shown. Below are representative samples of immunostained cells expressing the mutant forms of myc-tagged TFIIS. The relative strength of nuclear targeting by each mutant is given in the table at the bottom together with the structural role predicted for the substituted residue by Booth et al. (17). Targeting efficiency was assessed on the basis of the most common localization pattern observed for each mutant as well as the proportion of cells still exhibiting the predominantly nuclear distribution observed for wild-type TFIIS, and is expressed on a scale from no nuclear targeting (−) to wild-type levels (+++).

Figure 6

Expression of TFIIS mutant L28R in HeLa cells. (A) Immunoblot of myc-tagged TFIIS wild type (wt) and point mutants (1–6) from extracts of transfected HeLa cells probed with mAb 9E10. Mutants shown are lane 1, T37K; lane 2, L31R; lane 3, L28R; lane 4, L25K; lane 5, A9P; lane 6, A9D. (B) The effect of leptomycin B (LMB) on the cytoplasmic localization of hTFIIIS mutant L28R. HeLa cells expressing L28R were incubated in the presence (+) or absence (−) of LMB prior to immunostaining TFIIS with serum 36H. The distributions of overexpressed TFIIS mutant or wild-type polypeptides are shown in comparison with nuclear DAPI staining. (In comparison with 9E10, 36H does not contribute to detectable cytoplasmic immunostaining, rather endogenous TFIIS produces only faint background nuclear staining at the imaging exposures used here).

Figure 7

The region of TFIIS containing the LW motif mediates a protein–protein interaction. Pull-down assay of ³⁵S-labelled hIWS1 binding to immobilized GST-fusion proteins containing regions of TFIIS (indicated above the lanes). Graphical representation of this data is shown below in comparison with 20% of the labelled input protein and a pull-down using GST alone.