Neurospora crassa ro‐10 and ro‐11 genes encode novel proteins required for nuclear distribution

Abstract

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule‐associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro‐1, ro‐3 and ro‐4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro‐10 and ro‐11. ro‐10 and ro‐11 are non‐essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro‐10 and ro‐11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin‐related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro‐10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro‐11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.

Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

“…These results are very similar to the result from the dimorphic fungus Ustilago maydis in which the microtubule plusend localization of dynein is implicated in transporting endosomes from the microtubule plus end toward the minus end in hyphae (Lenz et al 2006). Consistently, dynein comets are more prominent in the absence of NUDF-interacting protein NUDE/RO11 in A. nidulans and Neurospora crassa (Minke et al 1999;Efimov 2003). Thus, while LIS1 homologs in filamentous fungi may be targeted to the microtubule plus end via mechanisms different from that of dynein's plus-end targeting (Efimov et al 2006), they are not required for dynein's plus-end localization, but instead may facilitate dynein's departure from the microtubule plus end.…”

LIS1—no more no less

“…The RO11 protein is known to participate in the cytoplasmic dynein pathway. 31 NUDE contains a coiled-coil domain that is present in several proteins including the mitotic phosphoprotein 43 (MP43) of Xenopus laevis, and a related Drosophila gene. 32 There are two mammalian homologs NUDEL and mNudE or rNudE, and they share 85% similarity in the coiled-coil domain.…”

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

“…Isolation and initial genetic characterizations of DHC mutant strains DHC mutant strains were isolated by employing a previously described genetic screen where mutations resulting in the loss of dynein/dynactin function were identified as partial suppressors of the cot-1 temperature-sensitive (cot-1 ts ) mutation (Plamann et al 1994;Bruno et al 1996;Tinsley et al 1996;Minke et al 1999). The N. crassa cot-1 gene encodes a serine/threonine protein kinase required for hyphal elongation (Yarden et al 1992).…”

“…The cot-1 ts mutation results in normal radial colony growth similar to wild-type strains at permissive temperatures (25°) and in small (,1 mm) colonies at restrictive temperatures (37°) (Supporting Information, Figure S1). Mutations resulting in a loss of dynein/ dynactin function partially suppress the cot-1 ts growth defect (Plamann et al 1994;Bruno et al 1996;Tinsley et al 1996;Minke et al 1999). In brief, 10 5 conidia from the cot-1 ts strain were suspended in 30 ml of cooled Vogel's minimal agar media (50°), plated, and incubated for 3-4 days at 37°.…”

“…The goal of this study was to examine the effects of DHC mutations where the DHC polypeptide is produced but has deficient function. Therefore, anti-DHC antibodies were used to screen by Western analysis the 290 ro-1 mutants for those that still appeared to produce full-length protein (Minke et al 1999(Minke et al , 2000Kumar et al 2000;Lee et al 2001). Seventy-six of the 290 ro-1 mutants produced what appeared to be full-length DHC protein.…”