A gene required for nuclear migration in Neurospora crassa codes for a protein with cysteine‐rich, LIM/RING‐like domains

Abstract

SummaryThe ro mutants of Neurospora crassa are defective in nuclear migration and hyphal morphogenesis. Several of the ro loci have recently been shown to encode components of the dynein/dynactin motor complex. Here we report on the cloning and characterization of the ro-2 gene which codes for a novel 80 kDa protein that has two Cys-rich motifs which resemble zinc-binding LIM or RING domains thought to mediate protein-protein interactions. RO2 also contains several potential binding sites for Src homology 3 (SH3) domains. The ro-2 B20 allele has a frameshift mutation within one of the Cys-rich domains which eliminates the C-terminal half of the open reading frame (ORF). Disruption of the ro-2 locus by repeat-induced point (RIP) mutation gave rise to progeny which have a nuclear migration defect, but which are also blocked in conidiation. The ability to assemble cytoplasmic microtubules and actin is maintained in ro-2 mutants, although subapical actin patches are more prominent. Based on these observations, the RO2 gene product is proposed to play a role in mediating interactions between components of the dynein/dynactin motor complex or in linking this complex to the nucleus or cytoskeleton.

A Dynactin Subunit with a Highly Conserved Cysteine-rich Motif Interacts Directly with Arp1

“…This gene from N. crassa is required for proper nuclear migration and hyphal elongation and was suggested to encode a dynein or dynactin subunit (21). Other N. crassa genes encoding subunits of either cytoplasmic dynein or dynactin have also been shown to produce similar phenotypes when disrupted (reviewed in Ref.…”

“…that ro-2 encoded a subunit of either protein complex (21), the absence of genetic interactions or biochemical data precluded a more definitive identification. Therefore, to characterize the 62-kDa vertebrate polypeptide as a dynactin subunit, we performed several biochemical and immunocytochemical assays.…”

“…Data base searches also identified ESTs with predicted cod-ing sequences for p62 homologs from mouse and Drosophila, as well as a predicted protein sequence for a Caenorhabditis elegans homolog generated through the genome project (21). The predicted sequences for human, mouse, Drosophila, C. elegans, and N. crassa are aligned in Fig.…”

“…The C. elegans sequence is a predicted open reading frame resulting from the genome project. The N. crassa sequence is from Vierula and Mais (21).…”

Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

“…Whereas during germination (the process in which a germ tube is formed from a round-shaped germ cell) in A. nidulans nudA mutant nuclei are not transported into the hyphal tube, the dynein heavy-chain mutants, such as dhc1, in the filamentous fungus Ashbya gossypii show a clustering of nuclei in the hyphal tips (2). In the N. crassa ropy class of mutants, with several genes of the dynein/dynactin complex, defects in nuclear positioning were also accompanied by morphological alterations (6,19,25,26). In contrast to the hyphal morphology of the wild type, ropy mutants show distorted morphologies indicative of a loss-of-growth directionality and an out-of-tip-focus Spitzenkörper (21).…”