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Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.Double-strand breaks (DSBs) in DNA are induced by ionizing radiation and by various chemical compounds, such as free radicals and methyl methanesulfonate (MMS). In addition, DSBs arise as intermediates during V(D)J rearrangement in differentiating lymphocytes, transposition events, and meiotic recombination in germ cells. Unrepaired DSBs often lead to cell death or contribute to the formation of chromosomal aberrations such as deletions and translocations. In eukaryotes, DSBs can be repaired via two main pathways: (i) recombinational repair, which is dependent on the presence of an intact duplicate DNA sequence, and (ii) end-to-end rejoining, which is based on rejoining of the two DNA ends (46). The available evidence suggests that both mechanisms are conserved in eukaryotes from yeasts to humans. However, the relative contributions of both mechanisms in repair of DSBs differ considerably between lower and higher eukaryotes. Repair of DSBs in mammals has been investigated with radiation-sensitive rodent cell lines. Several of these cell lines have defects in repairing radiation-induced DSBs and are also strongly impaired in V(D)J rearrangement. Further studies have revealed that these mutants have defects in the end-to-end rejoining pathway (reviewed in references 26, 47, and 63). Repair of DSBs at defined sites in the genome of mammalian cells also occurs more frequently by end-to-end rejoining than by homologous recombination (21, 33). The same observations have been made in experiments studying the fate of linear plasmid molecules in mammalian cells and Xenopus laevis oocytes (27,29,31). Therefore, DSBs in higher eukaryotes appear to be repaired primarily by end-to-end rejoining mechanisms.In lower eukaryotes, however, repair of DSBs occurs almost exclusively by...