The "8-kD" cytoplasmic dynein light chain is required for nuclear migration and for dynein heavy chain localization in Aspergillus nidulans

Abstract

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the "8-kD" cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.

Figure 1

Comparison of the growth at permissive and restrictive temperatures of a wild- type strain, nudG8, ΔnudA, and a nudG8/ΔnudA double mutant strain.

Figure 2

Sequence comparison of the A. nidulans 8-kD CDLC (NUDG) with other GenBank sequences. The deduced amino acid sequence of the 572-bp cDNA compared with Caenorhabditis elegans cosmid T26A5 sequence (U00043), Drosophila melanogaster CDLC1 (U32855), Homo sapiens CDLC1 (U32944), the Rattus norvegicus protein inhibitor of neuronal nitric oxide synthase (U66461), the 8-kD outer arm dynein light chain from Chlamydomonas reinhardii flagella (U19490), the T cell–stimulating antigen from the blood fluke Schistosoma mansoni (X98619), the Schistosoma mansoni DLC (U55992), and the DLC1 of Saccharomyces cerevisiae (U36468). The sequences were aligned with DNASTAR using the Clustal method with the PAM250 residue weight table. Residues that match the consensus sequence are shaded in black.

Figure 3

(A) Western blot of A. nidulans total protein stained with anti–NUDG antibody. The NUDG/CDLC antiserum recognized a single protein with an apparent molecular weight of 8-kD in cell-free extracts from a wild-type strain (GR5) grown at restrictive temperature (44°C), as indicated by the star. Under the same conditions, no 8-kD CDLC band was seen in the nudG8 mutant strain. (B) Western blot of A. nidulans total protein stained with anti–NUDA CDHC antibody.

Figure 4

Immunoprecipitation of proteins from a cell-free extract of A. nidulans grown at 44°C. The A. nidulans strains and the antibodies used for immunoprecipitation appear above the rows of blots. The antibodies used to detect NUDA and NUDC proteins in the blots are indicated to the right. The blots in the top row were stained with affinity purified A. nidulans NUDA/CDHC antibody and the bottom row were stained with A. nidulans NUDG/CDLC antiserum. From left to right, the seven columns of blots represent (from left) a control IP without antiserum, immunoprecipitation of a wild type extract with antibody against NUDA, immunoprecipitation of a wild-type extract with antibody against NUDG, immunoprecipitation of a nudA1 extract grown at restrictive temperature with antibody against NUDA, immunoprecipitation of the same extract with antibody against NUDG, immunoprecipitation of a nudG8 extract grown at restrictive temperature with antibody against NUDA, and immunoprecipitation of the same extract with antibody against NUDG.

Figure 5

Sucrose gradient sedimentation profiles of the NUDA and NUDG proteins. A sample from each of the top nine fractions from the gradient was subjected to SDS-PAGE and Western blotted with antibody against the nudA CDHC or the nudG 8-kD CDLC.

Figure 6

Immunofluorescence and Western blot analysis of the CDHC in wild-type and the nudG8 mutant strains. GR5 wild-type (WT) and nudG8 germlings grown at restrictive temperature (44°C) were stained with antibody against the NUDA CDHC protein. The cytoplasmic dynein heavy chain stains intensely at the tip of wild-type germ tubes. In contrast, there is little or no anti–CDHC antibody staining at the tip of the germ tube in the nudG8 mutant strain.