The role of Fis1p–Mdv1p interactions in mitochondrial fission complex assembly

Abstract

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)–like fold and a short NH2-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p–Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH2-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

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

“…Expression of Fis1 in fis1⌬ yeast restores WT morphology to ϳ80% of the level observed for wild type yeast. Consistent with earlier findings (17,33), Fis1-⌬N restores WT morphology to only 10% of the wild type level. A mixed morphology phenotype is seen for fis1⌬ yeast expressing Fis1-E78A, with only half of cells exhibiting WT morphology.…”

“…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.…”

“…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. 4, A and B).…”

“…We infer that the loss of Fis1/Dnm1 co-IP signal results from a loss of interactions with other protein(s) involved in this Fis1/Dnm1 complex. Candidates include Mdv1, which is thought to act as an adaptor between Fis1 and Dnm1 (7,11,33,43), and Caf4, which also binds Dnm1 and Fis1 (11,12), both of which have reduced binding to Fis1-E78A ( Fig. 3 and supplemental Fig.…”

Mitochondrial factors with dual roles in death and survival

“…The small outer mitochondrial membrane protein Fis1 recruits cytosolic Dnm1/Drp1, facilitating its ability to cycle on and off mitochondria. The interaction of yeast Fis1 and Dnm1/Drp1 is mediated by bridging molecules (Tieu and Nunnari, 2000;Cerveny and Jensen, 2003;Griffin et al, 2005;Karren et al, 2005;Naylor et al, 2006). The bridging factor Mdv1/Net2 is a WD repeat protein that has a partially redundant function with its closest homologue Caf4 (Griffin et al, 2005).…”