Evolutionarily conserved self-association properties of p150 CAF-1 proteins and their requirement for nucleosome assembly activity in vitro

The interaction of the conserved C-terminal third (Cter) of hp150 with its Xenopus counterpart in the two-hybrid screen raised two questions: (i) whether the interaction is direct and (ii) whether homodimerization or multimerization of p150 Cter can occur within the same species.

We first tested the interspecies interaction by co-expres sion in bacteria of histidine-tagged hp150 derivatives together with a glutathione S-transferase (GST)-tagged xp150 corresponding to the portion initially retrieved in our two-hybrid screen (amino acids 540–896) (Figure 2A, left). The GST–xp150 derivative could bind specifically to the hp150(620–938) peptide, but not to hp150(678–938), a peptide that is 58 amino acids shorter (Figure 2A, left, lanes 2, 3 and 5, 6). This interaction was stable up to 1 M salt extraction. Within the same species, the expression in bacteria of a combination of His₆ and GST hp150 derivatives (Figure 2A, middle) demonstrated self-interaction properties again requiring this stretch of 58 amino acids (Figure 2A, middle, lanes 2, 3 and 5, 6). These interactions were confirmed by a yeast two-hybrid assay (data not shown). We also tested the homo-interaction in Xenopus. The full-length His₆-tagged xp150 can interact with the GST–xp150(540–986) derivative but not with GST alone. This interaction is also stable up to 1 M salt extraction (Figure 2A, right, lanes 2, 3 and 5, 6). Further more, since aggregation of proteins could occur in GST pull-down assays, sedimentation analyses were performed with in vitro translated ³⁵S-labelled (not shown) and bacterially expressed human and Xenopus full-length p150 proteins. When physiological salt conditions were used in the gradients, the majority of the bacterially expressed human and Xenopus full-length p150 proteins sedimented at a similar position to the 258 kDa molecular weight standard (Figure 2B, WT hp150 and WT xp150, centred around fraction 17). Remarkably, an internal deletion restricted to 36 amino acids in the critical region of hp150, outside of the ED domain (residues 642–678), gave rise to a protein sedimenting as a monomeric 150 kDa form when run in parallel under the same conditions [Figure 2B Δ(642–678), fraction 13]. When gradients were performed in the presence of 2 M NaCl to disrupt p150– p150 interactions, the majority of wild-type hp150, hp150Δ(642–678) mutant and xp150 behaved as monomeric 150 kDa proteins (Figure 2B).

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Fig. 2. Inter- and intraspecies p150 dimerization: identification of a critical domain and role in chromatin assembly. (A) p150 self-association by GST pull-down assay. Left: GST–xp150(540–896) was co-expressed in bacteria (Studier et al., 1990) containing hp150(620–938) or hp150(678–938) tagged with His₆. Middle: His₆-tagged hp150(620–938) was co-expressed in bacteria with either GST–hp150(620–938) or GST–hp150(678–938). Right: equal volumes of separate bacterial extracts containing the full-length His₆-xp150 and GST–xp150(540–986) were mixed at 2 M NaCl; the salt concentration was lowered by sequential dilution to 350 mM NaCl, and glutathione–Sepharose resin was then added. Protein production and purification on glutathione–Sepharose resin were performed essentially as described (Moggs et al., 2000). Co-purification of His-tagged hp150 proteins was detected by western blot probed with anti-His₆. The xp150 proteins were detected using the specific anti-xp150 serum 566. I, input; B, bound fraction; B*, 1 M NaCl-resistant bound fraction. The positions of His₆-hp150(620–938) and His₆-hp150(678–938) are indicated. Asterisks mark degradation products. (B) Glycerol gradient sedimentation of p150 proteins. Bacterially produced recombinant proteins wild-type hp150, hp150Δ(642–678) and wild-type xp150 were sedimented on a linear 10–40% glycerol gradient. Every second fraction of the gradient was run on SDS–PAGE. The proteins were detected by the specific anti-hp150 and anti-xp150 sera on western blots. The sedimentation positions of the molecular weight standards for each gradient are indicated above the gel: BSA (66 kDa) and catalase (258 kDa). Arrows mark the mean position of the proteins. The salt concentrations used in the gradients are as indicated. (C) Chromatin assembly analysis by supercoiling in the presence of hp150Δ(642–678). pBscript control (–) and UV-irradiated (500 J/m²) (+) were incubated as in Figure 1D with S100 extract (–), or S100 extract complemented by HeLa nuclear extract (NE) or in vitro translated hp150 wild-type (lanes 5–6), hp150Δ(642–678) and xp150 wild type. The positions of the relaxed/nicked (Ir,II) and supercoiled DNA (I) are indicated.

Therefore, the self-association of the p150 CAF-1 subunit involves primarily dimeric forms, both in human and in Xenopus, and requires a conserved stretch of 36 amino acids, outside of the ED domain, in the C-terminal third of the proteins. Remarkably, searching through data banks for this motif did not retrieve any other protein, indicating that it is unique to p150. Based on these data, we then tested the functional importance of this domain in p150 using the in vitro assay for chromatin assembly coupled to repair of UV-damaged DNA as above. In vitro translated wild-type human and xp150 as well as the hp150Δ(642–678) mutant were used to complement the S100 extracts. Both full-length human and Xenopus p150 promoted nucleosome assembly onto damaged DNA as efficiently as a nuclear extract providing a functional CAF-1 complex (Figure 2C, lanes 4, 6 and 10). In contrast, the hp150Δ(642–678) mutant that does not homodimerize (Figure 2B) failed to complement the S100 extract (Figure 2C, lane 8). Thus, deletion of the 36 amino acid motif that was found critical for p150 self-association also abrogates p150 competence for chromatin assembly in vitro.

The largest subunit of human CAF-1 expressed in the early Xenopus embryo interacts with Xenopus p150 and arrests development

The discovery that a unique, conserved 36 amino acid stretch in p150 is required for homodimerization of the protein and for its chromatin assembly activity prompted us to consider the use of a dominant-negative strategy (Herskowitz, 1987) to study the requirements for CAF-1 in vivo. The Xenopus embryo and its rapid divisions at early stages (Kirschner et al., 1985) was an ideal in vivo system to test the importance of a factor potentially critical for coordinating chromatin assembly with DNA replication and repair. The RNA encoding the hp150 subunit was injected into one blastomere of a two-cell stage Xenopus embryo, and both the interaction with xp150 and its effect on development were examined. Immunoprecipitations using anti-xp150 or anti-hp150 antibodies were performed on extracts derived from embryos injected with hp150 RNA (Figure 3A). As shown in Figure 3B, when anti-xp150 antibodies were used in the immunoprecipitation all the detectable hp150 protein present in the embryo was found associated with xp150 (Figure 3B, compare lanes 2 and 3, hp150). Conversely, only a fraction of the xp150 present in the embryo was bound to hp150 (Figure 3B, lanes 4 and 5, xp150). This was a good indication that the amount of exogenously expressed protein was within the range of the endogenous counterpart and that hp150 was not overexpressed in our experiments. We also searched in these immunoprecipitates for proteins known to associate with hp150, such as PCNA (Shibahara and Stillman, 1999; Moggs et al., 2000) and acetylated histone H4 (Verreault et al., 1996). No detectable amounts of these factors were found associated with either human or Xenopus p150 proteins (Figure 3B, lanes 3 and 5, PCNA, AcK5-H4). We also verified whether other histone chaperones such as nucleoplasmin or N1/N2, previously shown to participate in nucleosome formation in Xenopus oocyte or egg extracts (Dilworth et al., 1987; Kleinschmidt et al., 1990), were bound to human or Xenopus p150. Under our experimental conditions, we did not find any significant co-precipitation of these proteins with either of the p150 proteins (Figure 3B, lanes 3 and 5, nucleoplasmin; and data not shown). Although we cannot formally exclude minor or transient interactions with other factors, the majority of the hp150 expressed in the early embryo was bound to the endogenous xp150.

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Fig. 3. Human p150 expressed in Xenopus embryos associates with Xenopus p150. (A) Scheme of the experiment: Xenopus embryos at the two-cell stage prepared as described in Vize et al. (1991) were injected into one blastomere using a nanoject injector (Drummond) with 1 ng of RNA encoding hp150 CAF-1. Embryos collected at the gastrula stage were processed for immunoprecipitation assay. (B) Immunoprecipitation of extracts from embryos injected with hp150 RNA using anti-xp150 antibody, anti-hp150 antibody or control antibody as indicated on top. Western blot of the different fractions: I, input; S, supernatant; P, pellet. hp150 and xp150 were detected by the polyclonal 573 and 566 antibodies, respectively; PCNA by the PC10 monoclonal antibody; nucleoplasmin by the monoclonal PAC35 antibody; and histone H4 acetylated at Lys5 by the polyclonal anti-H4AcK5 antibody.

We then analysed whether expression of hp150 in the early Xenopus embryo and its interspecies association with xp150 had any effect on development. The appearance of the protein was monitored by western blotting in parallel (Figure 4A, bottom). Control embryos, injected only with buffer or RNA encoding green fluorescent protein (GFP) as a cell lineage marker, developed normally (Figure 4A, buffer and GFP RNA). In the latter case, GFP fluorescence visible around the mid-blastula transition (MBT) was dispersed widely on one side of the embryo, but was virtually absent on the other (Figure 4A, GFP RNA), as previously reported (Zernicka-Goetz et al., 1996). In contrast, the injection of hp150 RNA severely affected embryo development, which did not reach the mid-blastula stage (Figure 4A, hp150 + GFP RNA). This arrest of Xenopus early development was obtained reproducibly when 1 ng of RNA encoding hp150 was injected (a usual amount for embryo injection) (Vize et al., 1991). However, amounts of RNA <100 pg were not effective, suggesting that a threshold in the expression of hp150 must be reached, probably to titrate the endogenous xp150 quantitatively in order to induce the developmental interference. Since these abnormal embryos were only detected when hp150 RNA was injected, we could exclude non-specific effects due to the microinjection itself or to exogenous RNA overexpression. Importantly, the timing of the effect, before the MBT, during a period of active division without significant transcription, together with the detection of large cells deriving from the injected side of the embryo (Figure 4A, see white arrowhead, Figure 4C) argues for a defect in cell cycle progression. For a closer examination of these aberrant large cells, embryos injected with buffer and hp150 + GFP RNAs were processed for cryosection at the early gastrula stage when GFP was detectable (Figure 4B). Strikingly, in the embryos co-injected with GFP and hp150 RNAs, 4′,6-diamidino-2-phenylindole (DAPI) staining was excluded from the area where GFP was detected (Figure 4B, top and bottom, hp150 + GFP RNA, white arrowheads) whereas a normal DAPI staining was observed in the embryos injected only with buffer. Immunofluorescence staining with the anti-hp150 monoclonal antibody (mAb1) confirmed that the distribution of hp150 overlapped with that of the GFP lineage marker (Figure 4B, bottom, GFP + hp150). We thus concluded that expression of hp150 was not compatible with the detection of normal nuclei at this developmental stage.

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Fig. 4. Human p150 expressed in Xenopus embryos specifically perturbs early development. Xenopus embryos were injected with RNA as in Figure 3A. (A) Development of embryos injected with: buffer, 2 ng of RNA encoding GFP (GFP) or a mixture of RNA encoding GFP and hp150 CAF-1, 1 ng each (hp150 + GFP) was followed at 23°C. Time after fertilization and corresponding developmental stages (Nieuwkoop and Faber, 1994) are indicated. Images of the embryos recorded at the indicated times after fertilization are presented as merges of the visible and GFP channels. The white arrowhead indicates the area of the embryo where the defect is first visible (pre-MBT, top right corner). The scale bar is indicated. For each stage, expression of hp150 in embryos injected with buffer (–) or with hp150 + GFP RNA (+) was monitored by western blotting (bottom) using the polyclonal 573 antibody. (B) Embryos injected with buffer and hp150 + GFP RNA were selected at early gastrulation stages and processed for cryosection and immunodetection. The injected RNAs or buffer are indicated on the left. Top: whole embryo sections, stained with DAPI to visualize DNA, were observed in bright field (left column) and appropriate filters for DAPI (DNA, middle) and GFP (right) fluorescence as indicated. Bottom: embryo cryosections were processed for immunofluorescence, and hp150 was detected using the mAb1 monoclonal antibody. DNA (revealed by DAPI), GFP and hp150 (Texas red) fluorescence were recorded with appropriate filters as indicated on the top. White arrowheads indicate areas of the section where GFP and hp150 are expressed but where DAPI staining is absent. The scale bar is indicated at the bottom. (C) Same pre-MBT Xenopus embryos as in (A). For each case, a higher magnification of the injected area is presented on the right. Scale bars are indicated at the bottom.

The 36 residues of p150 required for dimerization are critical to generate the negative interference in the embryo

The next step was to determine whether the domain of hp150 crucial for the dimerization was critical to generate the effects observed in the in vivo interference assay. Various truncated forms of hp150 were expressed in the Xenopus embryo (Figure 5A). Equivalent expression levels were verified by western blotting (data not shown) to compare the effects on embryonic development. Injection of RNA corresponding to a form of hp150 lacking the ED or KER box produced developmental defects comparable to those observed for wild-type hp150 (Figure 5A, WT, ΔED; and data not shown). Since the regions of hp150 containing the ED and KER boxes have been proposed to mediate interactions with histones (Kaufman et al., 1995), a simple histone titration appeared unlikely to explain the observed phenotype. This is in agreement with the results of the immunoprecipitation shown in Figure 3. Importantly, development was normal when the injected RNA encoded a hp150 mutant lacking the conserved C-terminal third of the protein [Figure 5A, (1–649)]. We thus expressed this latter region of hp150 in the embryo and found it sufficient to generate the abnormal phenotype [Figure 5A, (620–938)]. We next tested whether p150 mutants that cannot dimerize in vitro have an effect on early Xenopus development. Injection of the RNA coding for a truncated form of hp150 lacking the critical domain for self-association [Figure 5A, (678– 938)], as well as full-length hp150 lacking this internal domain [Figure 5A, Δ(642–678)], did not impair embryo development. These results are consistent with the interspecies association of the largest subunit of CAF-1 to mediate the arrest of early embryo development.

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Fig. 5. The dimerization domain of p150 is required to perturb Xenopus early development. (A) Xenopus embryos were injected with buffer or RNA encoding the full-length hp150 (WT) and truncated forms of hp150 RNA: (1–649), ΔED, (620–938), (678–938) and Δ(642–678), as indicated on the side. Images were recorded under a dissecting microscope at stages 9 and 36 of development (as judged from control embryos). The white arrowheads point to the area of the embryo where the defect is first visible. Scale bars are indicated. (B) Injections were as in (A) but with RNA encoding the full-length xp150 (WT) or xp150(540–896). On the right, a schematic representation of hp150 and xp150 proteins and a sequence alignment of dimerization domain for each species are shown.

Consistent with a dominant-negative effect, injection of mRNA coding for the xp150 Cter (amino acids 540–896) also impaired embryo development [Figure 5B, (540– 896)] whereas embryos injected with full-length xp150 RNA were not perturbed (Figure 5B, WT). The latter result allowed us to exclude a toxic effect simply due to a dosage variation of the CAF-1 p150 subunit. In conclusion, the in vivo interference with early Xenopus development mediated by hp150 or by truncations of xp150 protein required the presence of 36 residues in the C-terminal third of p150 that are critical for its dimerization and are conserved between the human and the Xenopus protein (Figure 5, right).

Co-expression of hp60 rescues the developmental defects mediated by expression of hp150

Remarkably, injection of RNA encoding hp60, the mid-subunit of CAF-1, did not alter embryonic development when injected alone (Figure 6, hp60), but could counteract the effect caused by expression of full-length hp150 (Figure 6A). In the same way, co-injection of hp60 RNA counteracted the effect caused by expression of hp150(620–938), which contains the p150 dimerization domain and is able to interact with hp60 in vitro, but is unable to support CAF-1 activity (Kaufman, 1995) (Figure 6B). This effect also argues once again for an interference specifically related to CAF-1. Furthermore, the fact that hp60 counteracts the interference caused by hp150(620–938) (Figure 6B) indicates that hp60 is acting by titrating out hp150 expressed in the embryo rather than by forming a functional complex.

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Fig. 6. Expression of hp60 counteracts the developmental perturbation induced by hp150 expression. (A) Embryos were injected with RNA encoding hp60 or hp150, or co-injected with hp60 and hp150 as indicated on the left (1 ng each). Images were recorded at stages 9 and 33 of development. Scale bars are indicated at the bottom. (B) Injections were as in (A) except that hp150(620–938) was injected.

Dimerization of human and Xenopus p150 subunits: a regulatory switch for CAF-1 activity?

By using the yeast two-hybrid system, in vitro binding assays and sedimentation analysis, we demonstrated that the human and Xenopus p150 CAF-1 subunits can homodimerize via their C-terminal domain (Figure 2A and B). This conserved property of p150 proteins requiring 36 homologous amino acids (Figures 1A and ​and5,5, right) explains interspecies interactions. Deletion of this region in hp150 abolishes the formation of p150 dimers (Figure 2A and B) and, most importantly, impairs its ability to promote chromatin assembly in vitro (Figure 2C). Furthermore, the interference assay shown in Figure 7C argues for the functional importance of a dimer of CAF-1–p150 to participate actively in nucleosome assembly. These data not only provide an explanation for the in vivo effect of hp150 expression but also have strong implications for the mechanism of CAF-1 function.

It is tempting to speculate that the p150 subunit alternates between homodimeric and dimeric forms and that these forms display different interaction properties. One possibility would be that the intrinsic component of the CAF-1 complex, i.e. the p60 and p48 proteins, would not bind equally well to these forms. In this context, it is interesting to note that based on immunoprecipitation experiments (Smith and Stillman, 1991), it was suggested that there might be a fraction of p150 that would not be associated with p60. Other interactions could also be modulated by the monomeric state of p150, with major consequences for the chromatin assembly reaction such as binding to PCNA and to histones H3 and H4. Future experiments to explore whether the monomeric p150 mutants are capable of binding to PCNA and to histones H3 and H4 will thus be very interesting and should provide major insight into the mechanism of CAF-1 function. In any case, a dynamic cycle between these possible p150-containing complexes could be used to regulate CAF-1 activity. The presence of DNA damage (Gaillard et al., 1996; Martini et al., 1998) or the rapid cell divisions of early embryogenesis that increase the requirement for CAF-1 may act on the equilibrium between p150 complexes. The regulation of this equilibrium could be accomplished by the binding of an as yet unidentified factor, or by post-translational modifications such as phosphorylation/dephosphorylation, since various phosphorylated forms of p150 do exist (Smith and Stillman, 1991; Marheineke and Krude, 1998; Martini et al., 1998; Keller and Krude, 2000). As a consequence, interfering with the rapid transition between p150 complexes would compromise CAF-1-dependent chromatin assembly activities in vivo.

Chromatin assembly assays

Analysis of DNA repair and chromatin assembly was performed as described (Gaillard et al., 1997). Briefly, circular pBscript plasmid either mock treated or UV irradiated (500 J/m²) was incubated with Xenopus embryo extract (HSE) (mock or xp150 depleted), S100 human cytosolic extract (Smith and Stillman, 1989) complemented or not by HeLa nuclear extract (Martini et al., 1998), in vitro translated or purified bacterially produced proteins or HSE, in the presence of [α-³²P]dCTP for 3 h at 23 (HSE) or 37°C (S100). The extent of supercoiling and DNA synthesis was examined by agarose gel electrophoresis followed by drying of the gel and exposure to X-ray film.

Immunological procedures

Polyclonal anti-xp150 antibody (serum 566) and anti-hp150 antibody (serum 573) were obtained after injections of purified GST–xp150(540– 896) and His₆-hp150(620–938) proteins in rabbits (AgroBio, Villeny). Both antibodies were affinity purified by incubating 200 µl of serum with 5–10 µg of purified GST or His₆ fusion protein present on nitrocellulose strips (Smith and Fisher, 1984).

For immunodepletion, 50 µl of HSE were mixed with an equal volume of anti-xp150 antibodies (566) bound to protein A–Sepharose for 30 min at 4°C. After centrifugation, the supernatant was subjected to a second round of depletion prior to its use for chromatin assembly reactions.

Embryo extracts were prepared by homogenization in buffer E [20 mM HEPES–KOH pH 7.5, 250 mM sucrose, 50 mM KCl, 2. 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml pepstatin and leupeptin] and centrifugation at 100 000 g for 30 min at 4°C. For immunoprecipitations, the cleared embryo extract was incubated with an equal volume of anti-xp150 affinity-purified antibody (566) or anti-hp150 affinity-purified antibody (573) for 1 h at 4°C. Protein A–Sepharose (Pharmacia Amersham Biotech) slurry (50 µl) equilibrated in buffer E was used to pull out the antibody.

Proteins were detected on western blots using the polyclonal anti-hp150 573 antibody (1/1000 dilution), the polyclonal anti-xp150 566 antibody (1/1000 dilution), the monoclonal anti-nucleoplasmin PAC35 antibody (1/500 dilution) (Dilworth et al., 1987), the monoclonal anti-PCNA antibody (PC10, DAKO, 1/500 dilution), the polyclonal anti-H4AcK5 antibody (1/500 dilution) (Turner and Fellows, 1989) and the anti-His₆ antibody (Santa Cruz, 1/000). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and the SuperSignal substrate detection kit (Pierce) were used for visualization.

For immunofluorescence, embryos were fixed overnight in 2% paraformaldehyde, 15% sucrose in 0.1× MBS [88 mM NaCl, 1 mM KCl, 0.41 mM CaCl₂, 0.33 mM Ca(NO₃)₂, 0.82 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM HEPES–KOH pH 7.4] solution, embedded in TissuTek (Sakura) and deep frozen in isopentane cooled by liquid nitrogen. Cryosections of 5 µm were prepared using a cryostat (Leica). The sections were adsorbed on superfrost plus slides and stored at –20°C. Human p150 was detected using mAb1 (1/100 dilution) (Smith and Stillman, 1991) and Texas red-coupled secondary antibody (Martini et al., 1998).

Recombinant proteins, GST pull-down

Recombinant proteins were co-expressed by transforming BL21 (DE3) bacteria (Studier et al., 1990) containing the pET30-hp150(620–938) plasmid or pET30-hp150(678–938) plasmid with pGEX4T-hp150(620– 938), pGEX4T-hp150(678–938) or pGEX4T-xp150(540–896). After induction with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 1.5 h at 30°C, cells were lysed in buffer A (20 mM Tris–HCl pH 8, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10 mg/ml pepstatin and leupeptin), sonicated briefly and the soluble fraction incubated with glutathione–Sepharose 4B resin (Pharmacia Amersham Biotech) for 1.5 h at 4°C. After extensive washes in buffer A, the resin was extracted with 1 M NaCl in buffer A. Total proteins bound to the resin were recovered by resuspending the washed resin directly in SDS–PAGE sample buffer (Laemmli, 1970). Bacterial extracts containing wild-type hp150, hp150Δ(642–678) and wild-type xp150 were produced as above. Proteins were purified over a nickel column (Pharmacia) according to the manufacturer’s recommendation and subsequently dialysed against HSE extraction buffer (Gaillard et al., 1997).

In vitro transcription–translation and glycerol gradients

Proteins were translated in vitro from the DNA constructs pβhp150/RN3, pβhp150Δ(642–678)/RN3 and pβxp150/RN3 using the TNT reticulocyte lysate coupled in vitro transcription–translation system (Promega) following the manufacturer’s instructions. Continuous 10–40% glycerol gradients in buffer G (20 mM Tris, 100 mM or 2 M NaCl, 0.025% NP-40, 2 mM EDTA) were poured into 5 ml tubes. A 50 µl aliquot of in vitro translated proteins or bacterial extract to which 150 µl of buffer G were added was loaded on the top of the gradients after a clearing spin of 10 min at 10 000 g at 4°C. Bovine serum albumin (BSA; 66 kDa) and catalase (258 kDa) were mixed and loaded in parallel in each gradient as sedimentation standards. The samples were centrifuged in an SW55 Beckman rotor at 45 000 r.p.m. for 18 h at 4°C. The refraction index was measured for each fraction collected (200 µl) to verify that gradients were identical.

DNA constructs

The plasmid pPK8 (Kaufman et al., 1995) was used to PCR clone the following fragments of hp150 (the first and last amino acids are indicated in parentheses): hp150(1–649) into pET30 vector (His₆ tag, Novagen); hp150(678–938) into pGEX4T-1 (GST tag, Pharmacia); and pET30 vectors. hp150(620–938) was excised from plasmid pNLX-hp150(620– 938) (see two-hybrid screen) and inserted into pGEX4T-1 and pET30 vector. The plasmids pPK8 and pED (Kaufman et al., 1995) were used to PCR clone full-length wild-type hp150, hp150(1–649) and hp150ΔED into the pβGFP/RN3 plasmid (Zernicka-Goetz et al., 1996). To produce hp150Δ(642–678), two PCR fragments were generated, one encoding hp150(1–642) and the second hp150(678–938). These PCR products were mixed and used as template for a second PCR with primers annealing 5′ of the first fragment and 3′ of the second. The resulting PCR product, hp150Δ(642–678), was cloned into pβGFP/RN3. The xp150(540–896) retrieved in our two-hybrid screen was cloned into pGEX4T-1. The complete xp150 cDNA was cloned as an EcoRI–NotI fragment into EcoRI–NotI-cleaved pβGFP/RN3P vector, generating pβxp150/RN3. Wild-type hp150, hp150Δ(642–678) and xp150 were subcloned from the pβ/RN3 plasmids into pET30 to generate His₆ fusions.