NUDF associates with NUDC at spindle pole bodies and at the cortex

interact and colocalize (Hirotsune et al., 1998). Although at first sight NUDF and NUDC localized at different sites in A. nidulans, the colocalization of their homologs in mammalian cells led to the investigation of the putative interaction between the Aspergillus proteins. An analysis of their association in a yeast two-hybrid assay was performed based on LexA and B42 fusions, respectively. It was intended to fuse NUDC to the B42 activation domain, but the fusion protein was not correctly expressed so that it could not be detected by Western hybridization (data not shown). Therefore, NUDC was fused to the LexA binding domain and this fusion was successfully detected by Western hybridization (data not shown). Association of LexA-NUDC with the B42-NUDF fusion was observed as indicated by expression of the LEU2 and lacZ reporter genes (Fig. 28B).

Furthermore, the domains should be specified by which the attachment between NUDF and NUDC is accomplished. Based on computer programs and the similarity to p23 (Garcia-Ranea et al., 2002), NUDC was predicted to consist of an N-terminal coiled-coil helix which replaces a large coiled-coil domain of mammalian NUDC, a p23-like central domain (a β-sandwich), and an unknown C-terminal domain of 83 amino acids (Fig. 28A).

NUDC-GFP (AGB241)

79 NUDF contains an N-terminal LisH dimerization motif followed by a coiled-coil helix and a large WD40 domain (a β-sheet propeller, Fig. 28A) (Kim et al., 2004; Tarricone et al., 2004). The parts of the open reading frames encoding these domains were fused to the respective two-hybrid domains and expressed in yeast in comparison to the full-length ORFs. The growth assay as well as the β-galactosidase filter assay revealed an interaction between the full-length NUDC and the WD40 domain of NUDF (Fig. 28B). In the growth assay, also a presumably false-positive interaction was detected between the coiled-coil domains of NUDF and NUDC through the expression of the more sensitive LEU2 reporter construct. No interactions were observed involving the separate p23 and C-terminal domain of NUDC, respectively, although all fusion proteins were detected by Western hybridization (data not shown). Thus, using two-hybrid analysis, the interacting domain of NUDC could not be identified.

A

B

Fig. 28 NUDF binds to NUDC via its WD40 domain.

(A) NUDF consists of a LisH dimerization motif followed by a coiled-coil helix (cc) and a WD40 domain.

NUDC was predicted to form an N-terminal coiled-coil helix, a p23 domain and a C-terminal domain of unknown structure. (B) In this yeast two-hybrid analysis, LexA-NUDC interacts with NUDF and B42-NUDFWD40 and induces LEU2 and lacZ reporter gene expression (EGY48 pRB1840 pME3246 pME3237 and EGY48 pRB1840 pME3246 pME3243). NUDF binding to the coiled-coil of NUDE (EGY48 pRB1840 pME2938 pME2939) was used as positive control (PC). NUDF and the empty prey vector (EGY48 pRB1840 pME2938 pJG4-5) were used as the negative control (NC). cc: coiled-coil. WD: WD40 domain.

To confirm the NUDF-NUDC interaction in vivo, the method of BiFC was used. For that purpose, the N-terminal and C-terminal halves of eYFP were fused to NUDF and NUDC, respectively, to observe bimolecular fluorescence complementation in case both fusion proteins were in close contact with each other. The fusions were expressed from the

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bidirectional niiA/niaD promoter and partially complemented the temperature-sensitive phenotypes when expressed in the nudC3 and nudF6 strain, respectively (AGB302, AGB303). These strains grew faster and produced more conidia than the temperature-sensitive parental strains at 42°C, but did not grow as fast as at 30°C which indicated partial functionality of the NUDF-nEYFP and NUDC-cEYFP fusions (data not shown).

Although the eYFP emission was low, numerous dots were detected along hyphae, but also at nuclei, which were labelled by constitutive expression of an mrfp::h2A fusion (Fig 29A).

Fluorescence was also detected at both poles of mitotic nuclei (Fig. 29A, lower row) showing that NUDF-NUDC interaction takes place at spindle pole bodies (SPBs) also during mitosis.

To clearly identify the colocalization of NUDC and NUDF at the spindle pole bodies, strain AGB335 was constructed harbouring the respective BiFC fusion construct and γ-tubulin (MIPA) fused to mRFP (Fig. 29B). In this strain again among several fainter fluorescent spots in the cytoplasm, a few prominent spots were observed, which could clearly be allocated to the mRFP-labelled spindle pole bodies. The SPBs often jerked in the cells, but were also found immobilized near the cortex in which case accurate superimposition with BiFC spots could be achieved best.

A B

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Fig. 29:NUDF binds to NUDC at spindle pole bodies.

(A) Bimolecular fluorescence complementation. Strain AGB303 expressing the nudF::neyfp and nudC::ceyfp fusions and the nuclear marker mrfp::h2A was cultivated in MM at 42°C for 8 h. Colocalization of NUDF and NUDC is shown through the fluorescence emission which can only be generated by the colocalization of the N- and C-terminal halves of eYFP (examples are indicated by arrowheads). Scale bars: 5 µm. (B) Bimolecular fluorescence complementation. Strain AGB335 expressing the BiFC constructs and the spindle pole body marker mipA::mrfp was grown in MM at 42°C for 8h. Scale bar: 5 µm.

To investigate whether the colocalization with SPBs could be confirmed for NUDC-GFP alone, a nudC::gfp fusion was introduced into a strain harbouring the mipA::mrfp fusion (AGB338, Fig. 30). In addition, nudC was not overexpressed, but expressed from the authentic promoter and was fused to the more stable gfp2-5 version. In fact, NUDC-GFP spots could be colocalized with MIPA-mRFP signals, even in mitotic nuclei (Fig. 30, lower row).

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Fig. 30: NUDC is localized at spindle pole bodies in A. nidulans.

In strain AGB338, NUDC-GFP colocalizes with γ-tubulin (MIPA) at spindle pole bodies. The strain was grown in glucose-containing MM at room temperature for 8 h. Scale bar: 5 µm.

These data demonstrate that NUDC and NUDF interact in A. nidulans. It was shown that the proteins colocalize at spindle pole bodies and at the cell cortex and that the association is mediated by the WD40 domain of NUDF. These findings correlate with observation in murine cells, where NUDC and LIS1 were colocalized at the microtubule organization center proposing fungal NUDC might be involved in spindle formation like hNUDC.

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