Deletion of a Novel F-Box Protein, MUS-10, in Neurospora crassa Leads to Altered Mitochondrial Morphology, Instability of mtDNA and Senescence

Abstract

While mitochondria are renowned for their role in energy production, they also perform several other integral functions within the cell. Thus, it is not surprising that mitochondrial dysfunction can negatively impact cell viability. Although mitochondria have received an increasing amount of attention in recent years, there is still relatively little information about how proper maintenance of mitochondria and its genomes is achieved. The Neurospora crassa mus-10 mutant was first identified through its increased sensitivity to methyl methanesulfonate (MMS) and was thus believed to be defective in some aspect of DNA repair. Here, we report that mus-10 harbors fragmented mitochondria and that it accumulates deletions in its mitochondrial DNA (mtDNA), suggesting that the mus-10 gene product is involved in mitochondrial maintenance. Interestingly, mus-10 begins to senesce shortly after deletions are visualized in its mtDNA. To uncover the function of MUS-

A uvs-5 Strain Is Deficient for a Mitofusin Gene Homologue, fzo1 , Involved in Maintenance of Long Life Span in Neurospora crassa

“…These age-matched progeny were initially characterized as wild-type, mus-10, and uvs-5 strains based on their sensitivities to UV light and MMS. Stocks of conidia with the appropriate phenotypes were confirmed by sequence analysis and then used in further experiments (12).…”

“…The mutagen sensitivities of N. crassa strains were investigated through spot tests as previously described (12). Conidia were suspended and adjusted to a concentration of 1 ϫ 10 6 conidia/ml.…”

“…DNA encoding the FLAG-tagged FZO1 protein was specifically integrated into the his-3 locus as described by Freitag et al and Kato et al (12,41). Primers KK09-ncu00436c-5 (5=-TCTAGAACATGAGCCAGGATTACTACCCG T-3=) and KK10-ncu00436c-3 (5=-GGATCCGTGGAAACCTCCAATAC CAGCA-3=) were used to amplify the full-length fzo1 gene (3,084 bp), using genomic DNA from wild-type N. crassa (74-OR23-1A) as the template.…”

“…Both the nd and sen strains accumulate deletions of mitochondrial DNA (mtDNA), though it is speculated that these arise through distinct mechanisms (8)(9)(10). Three UV-or mutagen-sensitive mutants of N. crassa, i.e., the uvs-4, uvs-5, and mus-10 mutants, also show a senescence phenotype accompanied by an accumulation of mtDNA deletions (11)(12)(13)(14).…”

“…Using a gene rescue approach, we found that the mus-10 gene encodes a protein that has an F-box domain. The physical interaction between MUS-10 and SCON-3 (an Skp1 homologue) and the necessity of the F-box domain for MUS-10 function suggested that MUS-10 is part of an SCF E3 ubiquitin ligase complex and functions in the degradation of an unknown target protein(s) (12). Thus, MUS-10 may act in a ubiquitin-proteasome pathway to regulate turnover of proteins required for maintenance of mitochondria and the life span (12).…”