Roles of NUDE and NUDF proteins of Aspergillus nidulans: insights from intracellular localization and overexpression effects

Abstract

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes ("comets") that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudE gene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditional nudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF and nudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between the nudF and apsA genes suggests a role for NUDF at the cell cortex.

Figure 1

Deletion of the nudE gene does not abolish the GFP::NUDF fusion localization to comet-like structures in A. nidulans hyphae. (A) The control GFP::nudF strain grown on the strongly inducing threonine medium. Despite the bright background, the comets can be seen in the video near the tip. (B) Hyphae of the GFP::nudF; ΔnudE strain were grown as described in A. Video shows the tip of the top hypha with several comets clearly visible. (C) The control GFP::nudF strain grown on threonine plus glucose to bring down the expression of the fusion. This is a region ∼200 μm away from the hyphal tip. The comets are clearly seen in the video. The significance of a structure in the center is not clear. (D) Typical example of a hyphal tip of the GFP::nudF; ΔnudE strain grown as described in C. The tips of the GFP::nudF strain looked the same. Bar, 5 μm.

Figure 2

Deletion of the nudE gene does not eliminate the comets of GFP::NUDA, but changes their behavior. Hyphae were grown under identical conditions on the strongly inducing threonine medium as for Figure 1, A and B. (A) Hyphal tips of the control GFP::nudA strain. The intensities in Video A1 were adjusted to brighten the image. Exposure for A2 was 0.6 s. (B) Hyphal tips of the GFP::nudA; ΔnudE strain. (C) An example of specks of GFP::NUDA in the internal hyphal region of the GFP::nudA strain. Video shows that the specks are immobile or bounce randomly, whereas a comet moves from left to right. The exposure time for this image and video was 0.6 s. Bar, 5 μm.

Figure 3

(A) Biological activities of the GFP-tagged NUDE protein variants. A nudE deletion strain (SF2-9) and the nudF7 ts mutant (XX21) were transformed with indicated genes in the multicopy vector pAid. Transformants were grown for 3 d on complete medium (YAG) without or with 0.6 M KCl at 43°C, which is a restrictive temperature for the nudF7 mutant. The fawn color of conidia in the ΔnudE strain is similar to the brownish color of nonconidiating mycelium. The color of conidia in the nudF7 strain is yellow. (B) GFP::NUDE variants are expressed at similar levels. Total protein extracts were made from a ΔnudE strain transformed with indicated genes in the pAid vector. Two different transformants were used for each GFP::NUDE variant. Proteins (12 μg/lane) were separated on a 10% SDS-PAGE and immunoblotted with an anti-GFP antibody. The bottom panel is Ponceau S staining of the membrane after protein transfer.

Figure 4

GFP-tagged NUDE and NUDE-C localize to comets, whereas NUDE-N distributes uniformly throughout the cytoplasm. A ΔnudE strain was transformed with pAid::GFP::nudE, pAid::GFP::nudE-N, and pAid::GFP::nudE-C (Figure 3A) and transformants were grown on a threonine medium as described for Figures 1, A and B, and 2. (A) Full-length GFP::NUDE fusion. The brightness of the image was slightly increased to show faint comets far from the tip. The video shows comets moving in both directions parallel to the hypha axis. The background fluorescence inside this hypha is typical for this fusion and never was as bright as in C or B. (B) GFP::NUDE-N. This is an example of GFP signal variability among individual hyphae. All four hyphae are in the same focal plane. (C) GFP::NUDE-C. The video shows that many comets move at an angle to the hypha axis. Bar, 10 μm.

Figure 5

Specks of the GFP-tagged full-length NUDE as observed after integration of several copies of the alcA(p)::GFP*:: nudE gene at the nudE locus. The intensities of images and time-lapse series were adjusted for reproduction purposes. As a result, very bright specks show as round objects instead of sharp dots. (A) Cells were grown in liquid M-glycerol medium for 2 d at 28°C. Ethanol was added to 1.7% (vol/vol) and the image and time series were taken 5 h later. Differential interference contrast image is shown below. The video shows that the specks move in a jerky way and in all directions. (B) Cells were grown as described in A. Benomyl was added to 4 μg/ml together with ethanol, and the image and time series were taken 4 h later. The video shows almost complete lack of movements. (C) In old hyphae, the specks are often imbedded into cables. The cells were grown for 2 d at 26°C on an agar pad of M-glycerol plus ethanol. The image was taken from the crowded area inside the colony. The movements in such areas were rare and were limited to isolated specks outside the cables. Differential interference contrast image is shown to the right. Bar, 5 μm.

Figure 6

(A) Total suppression of the nudE gene deletion by multiple copies of the nudF gene. A strain with the deleted nudE gene (SF2-9-9) was transformed with the nudF and nudE genes in the multicopy vector pAid and with the empty vector. Transformants were grown on complete medium (YAG) for 3 d. The strain produces conidia of wild-type color (dark green). (B) NUDF protein level is not affected by the deletion of the nudE gene and increases ∼10-fold after transformation with the pAid::nudF plasmid. Total protein extracts were made from the transformants shown in A as well as from a wild-type strain (GR5) transformed with pAid. Proteins were separated on a 4–20% SDS-PAGE and immunoblotted with an anti-NUDF antibody. Equal amounts (3.3 μg) of protein were loaded in the first four lanes, followed by serial dilutions of the ΔnudE[pAid::nudF] extract. The uppermost band is the full-length NUDF protein (49 kDa). The ∼30-kDa band enriched in the NUDF-overexpressing extract is a NUDF breakdown product. The lowest band is nonspecific and demonstrates equal loading.

Figure 7

Interactions between the nudF gene and nudC, nudA, apsA genes. Shown are A. nidulans mutants transformed with multicopy plasmids and grown for 3 d on plates with YAG or YAG plus 0.6 M KCl. The color of conidia is bright green in the nudA1 strain and yellow in other strains. (A) pAid::nudF suppresses the ts nudC3 mutant (strains C3y-3), whereas pAid::nudF6 inhibits it. (B) pAid::nudF inhibits the ts nudA1 mutant (strain XX3). (C) pAid::nudF inhibits the apsA5 mutant (strain apsA5). The strain is not affected by the temperature or KCl, and transformants look the same under all conditions. The apsA1 transformants also looked the same. The top colony is a wild-type control and is the nudF7 mutant (strain XX21) transformed with pAid::nudF. (D) Multiple copies of the apsA gene inhibit the ts nudC3 mutant (strains C3y-3) under partially restrictive conditions.

Figure 8

Summary of the effects of multiple copies of different genes on different A. nidulans mutants described herein and in the previous work (Efimov and Morris, 2000). The results with the truncated and chimeric nudE genes and GFP::nudE fusions are not shown (Figure 1 in Efimov and Morris, 2000; Figure 3A in this work). That multiple copies of the nudF gene suppress the nudC3 mutation was first reported by Xiang et al. (1995a). Each strain was transformed with a multicopy plasmid carrying the indicated gene, and transformants were compared with the same strain transformed with the empty vector. All mutants are conditional (temperature sensitive) except for the apsA, apsB and deletion mutants. Bigger symbols represent a stronger effect. Untested combinations are left blank. Notes: a–multicopy plasmids used to transform SRF30 (ΔapsA) strain carry argB as a selective marker, while the pyrG gene was used as a selective marker for transformation of all other strains; b–the only effect is a slight improvement in conidiation at 32°C, YAG; c–inhibition is observed under all conditions, but is most obvious at 37°C; d–slight improvement in conidiation at 32°C, YAG; e–slight improvement in conidiation at 43°C, YAGK; f–the inhibitory effect is subtle and observed in a narrow range of semirestrictive conditions (43°C, YAGK); g–no effect at 32°C (YAG or YAGK) and at 43°C, YAG, inhibition at 37°C (YAG or YAGK) and at 43°C, YAGK.