The WD repeat protein, Mdv1p, functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission

Abstract

Yeast mitochondrial fission is a multistep process during which the dynamin-related GTPase, Dnm1p, assembles into punctate structures that associate with the outer mitochondrial membrane and mediate mitochondrial division. Steps in the Dnm1p-dependent process of fission are regulated by the actions of the WD repeat protein, Mdv1p, and the mitochondrial outer membrane protein, Fis1p. Our previous studies suggested a model where Mdv1p functions to regulate fission at a post-Dnm1p assembly step and Fis1p functions at two distinct steps, at an early point, to regulate Dnm1p assembly, and later, together with Mdv1p, to facilitate Dnm1p-dependent mitochondrial fission. To test this model, we have examined the physical and functional relationship between Mdv1p and Fis1p and present genetic, biochemical, and two-hybrid data indicating that a Fis1p–Mdv1p complex is required to regulate mitochondrial fission. To further define the role of Mdv1p in fission, we examined the structural features of Mdv1p required for its interactions with Dnm1p and Fis1p. Data from two-hybrid analyses and GFP-tagged domains of Mdv1p indicate that it contains two functionally distinct domains that enable it to function as a molecular adaptor to regulate sequential interactions between Dnm1p and Fis1p and catalyze a rate-limiting step in mitochondrial fission.

A Targeted Mutation Identified through pK Measurements Indicates a Postrecruitment Role for Fis1 in Yeast Mitochondrial Fission

“…In GST pull-down experiments, the Fis1 cytosolic domain tagged with GST (Fis1⌬TM-GST) bound well to yeast-expressed MBP-Mdv1 (Fig. 3, A and B), demonstrating a direct physical interaction between Fis1 and fulllength Mdv1 that is consistent with previous co-IP, yeast twohybrid, and GST pull-down studies (15,16,33,35). This robust pull-down was almost completely lost for Fis1-E78A (Fig.…”

“…E78A Modestly Affects Mdv1 Mitochondrial LocalizationWe next determined how Fis1-E78A affects the localization of Mdv1 and Dnm1, which normally co-localize on the mitochondrial surface in distinct punctate structures, or foci, that mark future scission sites (15,35,44,45). As in previous findings (17,33), the absence of Fis1 ablated Mdv1 mitochondrial localization; 85% of wild type cells show punctate mitochondrial Mdv1, whereas 99% of fis1⌬ cells show cytoplasmic Mdv1 (Fig.…”

“…Fis1 has been reported to interact with both the fission adaptor Mdv1 (15,16,18,35) and the fission mechanoenzyme Dnm1 (16), although the significance of the Fis1/Dnm1 interaction is not as firmly established as the Fis1/Mdv1 interaction (36,37). After determining that the E78A substitution has little effect on Fis1 structure by NMR, its folding by chemical denaturation, its propensity to form dimers (38), or its ability to bind membranes (16) (supplemental Fig.…”

Mitochondrial factors with dual roles in death and survival

“…Yeast Fis1 (18 kDa) is slightly smaller than Bcl-2 family proteins (about the size of viral Bcl-2 proteins). It localizes to the outer mitochondrial membrane via its C-terminal hydrophobic-basic domain with its Nterminus in the cytosol, similar to Bcl-2 (Shaw and Nunnari, 2002;Tieu et al, 2002). Unlike Bcl-2, the three-dimensional structures of yeast, human and mouse Fis1, solved by three different groups, revealed that Fis1 is a tetratricho-peptide repeat (TPR) protein that is most superimposable with one of the TPR motifs in the peroxisomal protein Pex5, but is perhaps the most similar in overall structure and size to the mitochondrial import protein Tom20 (Abe et al, 2000;Suzuki et al, 2003;Dohm et al, 2004).…”

Mitochondrial fission proteins regulate programmed cell death in yeast

“…Dnm1 was reported to interact with the WD40 repeat domain of Mdv1/Net2 and with the Fis1 protein (Tieu and Nunnari 2000;Cerveny et al 2001;Tieu et al 2002;Cerveny and Jensen 2003;Yoon et al 2003). There are no obvious homologs of Mdv1/Net2 in mammals (outside the WD40 repeat domain), but Fis1 is highly conserved from plants to humans (Mozdy et al 2000;Tieu and Nunnari 2000).…”