Nonredundant Roles of Mitochondria-associated F-Box Proteins Mfb1 and Mdm30 in Maintenance of Mitochondrial Morphology in Yeast

Abstract

Mitochondria constantly fuse and divide to adapt organellar morphology to the cell’s ever-changing physiological conditions. Little is known about the molecular mechanisms regulating mitochondrial dynamics. F-box proteins are subunits of both Skp1-Cullin-F-box (SCF) ubiquitin ligases and non-SCF complexes that regulate a large number of cellular processes. Here, we analyzed the roles of two yeast F-box proteins, Mfb1 and Mdm30, in mitochondrial dynamics. Mfb1 is a novel mitochondria-associated F-box protein. Mitochondria in mutants lacking Mfb1 are fusion competent, but they form aberrant aggregates of interconnected tubules. In contrast, mitochondria in mutants lacking Mdm30 are highly fragmented due to a defect in mitochondrial fusion. Fragmented mitochondria are docked but nonfused in Δmdm30 cells. Mitochondrial fusion is also blocked during sporulation of homozygous diploid mutants lacking Mdm30, leading to a mitochondrial inheritance defect in ascospores. Mfb1 and Mdm30 exert nonredundant functions and likely have different target proteins. Because defects in F-box protein mutants could not be mimicked by depletion of SCF complex and proteasome core subunits, additional yet unknown factors are likely involved in regulating mitochondrial dynamics. We propose that mitochondria-associated F-box proteins Mfb1 and Mdm30 are key components of a complex machinery that regulates mitochondrial dynamics throughout yeast’s entire life cycle.

Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

“…The remaining 30% were partitioned between those scored as having fused, fizzed, and aggregated mitochondria (see ex-amples in Figure 1A). In agreement with previous reports (Fritz et al, 2003;Dürr et al, 2006;Escobar-Henriques et al, 2006), among mdm30⌬ cells almost 70% displayed aggregated mitochondria ( Figure 1C, vector transformation). Reintroducing MDM30 into mdm30⌬ cells (MDM30-HA), under a constitutive TEF promoter, restored mitochondrial morphology to a distribution similar to wild-type cells and completely reversed the marked increase in aggregated mitochondria seen in mdm30⌬ cells ( Figure 1C).…”

“…Despite the requirement for the Mdm30p F-box in Fzo1p degradation found in Escobar-Henriques et al, the same group found that Mdm30 lacking its F-box overexpressed from the CUP promoter surprisingly restored normal mitochondrial morphology in mdm30⌬ cells (Dürr et al, 2006). In contrast, we find that, in these cells, expression of F-box mutants of Mdm30p from either the TEF or MDM30 promoter results in a failure to reverse the abnormal aggregated mitochondrial morphology seen in mdm30⌬ cells, a finding confirmed by the failure to restore growth on a nonfermentable carbon source (glycerol).…”

“…A number of reports have provided indirect links between the UPS and mitochondria (Fisk and Yaffe, 1999;Sutovsky et al, 1999;Hitchcock et al, 2003;Peng et al, 2003;Thompson et al, 2003;Rinaldi et al, 2004;Altmann and Westermann, 2005;Dürr et al, 2006). The best evidence so far for involvement of the UPS in directly regulating mitochondrial outer membrane proteins is in mammals where a specific E3, MARCHV/ MITOL, is implicated in ubiquitylating two components of Table S1) by treating with CHX.…”

“…Loss of mitochondrial fusion has been shown to manifest itself as a failure of mdm30⌬ cells to respire properly and a decreased capacity to grow on media containing only a nonfermentable carbon source (Fritz et al, 2003;Dürr et al, 2006). Strains from Figure 1C were therefore tested for growth on selective media containing either dextrose (fermentable) or glycerol (nonfermentable) as the sole carbon source ( Figure 1D).…”

“…The importance of Mdm30p in mitochondrial function is underscored by the finding that deletion of MDM30 abrogates mitochondrial fusion and leads to aggregated mitochondria (Dürr et al, 2006; see examples in Figure 1A) and consequently to defective mitochondrial DNA inheritance and a failure to respire (Fritz et al, 2003). However, the mechanism by which Mdm30p promotes Fzo1p degradation has not been elucidated.…”