Efimov VP , Morris NR .

5RO1GM52309-03 NIGMS NIH HHS , R01 GM052309 NIGMS NIH HHS

	Figure 1. (A) Suppression of nud mutants by extra copies of the nudE and nudF6 genes. The indicated mutants (all are conditional, temperature sensitive) were transformed with either the empty vector pAid or pAid clones bearing nudE and nudF6 genes (plasmids recovered in the multicopy suppressor screen) and grown at 32°C. Transformants were gridded on YAG plates with or without KCl and incubated at 43°C or 32°C for 2 d. All strains have different color of conidia: yellow for nudF7, green (wild-type) for nudF6, chartreuse for nudA1, and white for nudC3. The intensity of the colony color is proportional to the number of conidia produced. Four independent transformants of the nudF7 mutant are shown for each plasmid to demonstrate reproducibility of phenotypes. (B) Complementation of the nudE deletion and the nudF7 mutant by extra copies of nudE, the nudE NH2-terminal domain, and nudE chimeras carrying coiled-coil regions from human and frog proteins, respectively. Strains were transformed with nudE variants in pAid vector and grown at 43°C. Numbers refer to amino acid residues of NUDE protein expressed by the constructs (see Fig. 3 for detailed amino acid sequences). Due to the presence of a fawn color marker, conidiating and unconidiating ΔnudE colonies have very similar colors at this temperature.
	Figure 3. (A) Homology between A. nidulans NUDE protein and N. crassa RO11 protein. Shaded residues are predicted to form a coiled-coil structure. The probabilities of coiled-coil formation are 0.9–1 for the NH2-terminal region and 0.7–0.8 for the central region. Probabilities of coiled-coil formation were calculated using program COILS version 2.1 (MTIDK matrix, unweighted a and d positions, window = 28) at http://www.ch. embnet.org/software/COILS_form.html (Lupas et al. 1991). Assigning 2.5-fold weights to a and d positions slightly changes the probabilities at coiled-coil ends and increases probability for the central coiled-coil to 0.9–1. (B) Alignment between coiled-coil regions of an ORF predicted from human EST with GenBank accession number AA424443, X. laevis MP43 (accession number U95097), A. nidulans NUDE (accession number AF085679), and N. crassa RO11 (accession number AF015560). The underlined residues are predicted to form coiled-coil structure with probabilities >0.9. The regions between arrowheads were used in sequence exchange experiments to test if human or X. laevis coiled-coil regions can substitute for the NUDE coiled-coil. The percentages of identical (similar) residues for the underlined coiled-coil regions are 69% (82%) between NUDE and RO11, 38% (51%) between NUDE and AA424443, 39% (51%) between NUDE and MP43, 72% (83%) between AA424443 and MP43, 40% (52%) between RO11 and MP43, 40% (52%) between RO11 and AA424443.
	Figure 2. Extra copies of the nudE gene do not change the level of NUDF protein in the nudF7 mutant. Total protein extracts were prepared from nudF7 and nudA1 mutants carrying indicated plasmids and NUDF protein levels were analyzed by Western blotting with the anti-NUDF antibody. Strains were grown at 43°C in YG medium. A nudA1 mutant was included as a control of the wild-type level of NUDF. The amount of NUDF protein in the nudF6 mutant is less than in nudF7 (Xiang et al. 1995a).
	Figure 4. NUDE and NUDF proteins interact in a two-hybrid system. S. cerevisiae strain AH109 was transformed with pairwise combinations of plasmids expressing indicated proteins as fusions with either the Gal4p DNA binding (Gal4pDBD) or Gal4p activating (Gal4pAD) domains. NUDE, AA424443 and MP43 refer to the NH2-terminal domains of A. nidulans NUDE protein (residues 1–195) and corresponding fusions with human and X. laevis homologues, respectively (Fig. 3 B). For each pair of plasmids, growth on three media is shown (from left to right): SD/-Leu/-Trp/-His; SD/-Leu/-Trp/-His with 3 mM 3-AT; SD/-Leu/-Trp/-Ade. The first two media select for the expression of the HIS3 reporter gene, while the third medium selects for the expression of the ADE2 reporter gene. Growth in the absence of histidine or adenine is expected to result from interactions between proteins encoded by plasmids. Note that Gal4pDBD fusions with the coiled-coil domains activate the HIS3 gene expression in the absence of any interactions. 3 mM 3-AT (3-amino-1,2,4,-triazole), a competitive inhibitor of the HIS3 gene product, suppresses the resulting background growth.
	Figure 5. NUDE protein tagged with VSV-G epitopes coprecipitates with NUDF in A. nidulans protein extracts. (A) Total protein extracts from four independently isolated NUDE::(VSV-G)6 strains (#8, 12, 15, 17) and the parent strain with the wild-type nudE gene were analyzed by SDS-PAGE (4–20%) and immunoblotting with P5D4 antibody (left). Ponceau S staining of the membrane after protein transfer is shown on the right. (B) Same samples as in A (tagged strain #17 and untagged control) after better separation on a 10% SDS-PAGE. (C) The left panel shows immunoblotting of proteins precipitated by P5D4 antibodies coupled to Sepharose beads from nudE::(VSV-G)6 and nudE+ extracts with P5D4 and anti-NUDF antibodies. The right panel shows silver staining of the same samples.

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