In this report, we use in vitro pull-down assays to test protein-protein interactions thought to be essential for fission of the mitochondrial outer membrane and peroxisomes of S. cerevisiae. We find that Dnm1 and Mdv1 bind directly to Fis1; Mdv1 binds via its NTE domain and does so independently of its coiled-coil domain, its WD repeat domain, or other proteins. Dnm1 binds directly into the concave surface of the Fis1 TPR-like domain and does so independently of Mdv1. The Fis1 arm appears to regulate access to the Dnm1 binding site ().
Autoinhibition model for Fis1 binding of Dnm1
Our Fis1 mutagenesis data on Dnm1 binding suggests an interaction between these two proteins that does not share a large buried surface area. The disruptive mutants of Fis1 lie in a narrow groove of the concave surface that is primarily hydrophobic (). Point mutants in either α-helix 2 (Q40A and N44A) or α-helix 6 (Q112E) that lie adjacent to α-helix 4 did not affect Dnm1 binding (). This finding is consistent with binding by a part of Dnm1 in extended conformation, such as the N or C terminus or an unstructured region between domains. Similar interactions are observed in other TPR domain proteins. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70, whereas the TPR2A domain of Hop binds the C-terminal pentapeptide of Hsp90 (51
). Also, the TPR domain of Pex5, a protein involved in peroxisomal targeting, recognizes a C-terminal tripeptide motif that is in extended conformation (52
). Identifying the Dnm1 residues important for binding to Fis1 will be the focus of future experiments.
Our results indicate that Mdv1 does not bind into the conserved surface of the Fis1 TPR-like domain. However, we cannot rule out that this surface mediates interactions with the C-terminal, WD repeat domain of Mdv1, which is not present in our constructs. Yeast two-hybrid data conflict on whether this interaction is possible (38
). Mdv1 is found only in fungi (40
), so we tested residues Gln28
, and Asn44
because they are more conserved in fungi (supplemental Fig. 1
). However, point mutants of these residues did not affect Mdv1 binding (). Based on sequence considerations, Fis1 residues on the convex side of the TPR-like domain between α1 and α2 are more conserved in fungi than mammals. Future studies will test the role of these Fis1 residues (Arg42
) in Mdv1 binding.
Given the new information presented in this study, previous in vivo
observations may be better understood. Several laboratories have reported interactions between Fis1 and Mdv1 by cytological or yeast two-hybrid experiments (25
). However, many attempts to observe an interaction between Fis1 and Dnm1 by yeast two-hybrid analyses have not revealed this interaction, although a recent report that budding yeast Fis1 and Dnm1 co-immunoprecipitate suggests that a direct interaction is possible (42
). This discrepancy may now be resolved in light of the present work, which suggests that a likely explanation for the lack of the yeast two-hybrid interaction may arise from the autoinhibition of Fis1 by its N-terminal arm.
Fis1 autoinhibition of Dnm1 binding may be relieved in the presence of Mdv1. We attempted to test this possibility with our constructs in a competition assay but did not observe an increase in Fis1-Dnm1 interaction upon increasing concentrations of Mdv1ΔWD (supplemental Fig. 4
). These data suggest that the NTE and CC domains are insufficient to enhance Dnm1 binding to Fis1. Perhaps full-length Mdv1 or another protein is necessary for relieving the autoinhibition of Fis1. Alternatively, it may be the presence of the mitochondrial outer membrane or a covalent modification, such as phosphorylation. Intriguingly, the Dbl homology domain of the proto-oncogene Vav also contains an N-terminal arm that inhibits Rho GTPase activation, and this inhibition is relieved by tyrosine phosphorylation that exposes the GTPase interaction surface (48
). Fis1 contains many putative phosphorylation sites, but whether this is important in regulating Fis1 activity in mitochondrial dynamics has not been reported to our knowledge.
The loss of the Fis1 arm in vivo
alters mitochondrial morphology (40
), which appeared to arise from failed recruitment of Mdv1 to mitochondria, and subsequently has been shown to be rescued by Mdv1 overexpression (41
). Our data are consistent with these physiological results, since they suggest that the Fis1 arm enhances the Mdv1 interaction. In the absence of this arm, we observe a decrease in binding to both Mdv1 constructs (). Thus, we think the reported loss of Mdv1 recruitment with ΔN-Fis1 in vivo
arises from decreased binding between Mdv1 and ΔN-Fis1 that we detect in vitro
. This decrease in affinity appears small enough to be rescued by Mdv1 overexpression in vivo
as reported (41
Our results may also help to explain the aberrant mitochondrial morphology of a temperature-sensitive allele of Fis1 that arises from mutations in three residues (E78D/I85T/Y88H) that lie in α-helix 4 of the TPR-like domain (41
). This temperature-sensitive phenotype is suppressed by either Mdv1 overexpression or a point mutant in the CC domain of Mdv1, implying a direct interaction between the conserved surface of Fis1 and the CC domain of Mdv1 (41
). Our mutagenesis data suggests another possible interpretation; perhaps this Fis1 triple mutant affects Dnm1 binding that results in aberrant morphology. In this situation, overexpression of Mdv1 might then be expected to stabilize Dnm1 on the Fis1 surface due to the reported interaction between the WD repeat domain of Mdv1 and Dnm1 (25
). Additionally, the point mutation in the CC domain may serve to relax an autoinhibitory ability of the Mdv1 protein. Consistent with this possibility, overexpression of an Mdv1 construct containing both NTE and CC domains inhibits mitochondrial fission (39
A direct interaction between yeast Fis1 and Dnm1 has implications for the mammalian orthologs, hFis1 and hDrp1. Previous studies show that hFis1 and hDrp1 are in close proximity by fluorescence resonance energy transfer and that these two proteins co-immunoprecipitate together (27
). We predict here that these results arise from a direct interaction between these proteins in a manner similar to the yeast molecules. Whereas recombinant human Fis1 and Drp1 were tested for a stable interaction that was not found (30
), perhaps human Fis1 is also autoinhibited by its own N-terminal arm in a manner similar to the yeast molecule. Two lines of evidence support this speculation. First, an hFis1 construct lacking its first 20 residues co-immunoprecipitates with hDrp1 to a much greater extent than Fis1 with a native N terminus (43
). Second, structural studies on the human and mouse Fis1 molecules suggest that such autoinhibition may be possible, although the mammal Fis1 arm is eight residues shorter than the yeast arm. The mouse Fis1 solution structure reveals the Fis1 arm blocking access to the TPR-like domain,5
whereas the human Fis1 solution structure allows access (45
). These differences speak to the dynamic nature of the Fis1 arm, since the amino acid sequences only differ by eight mostly conserved substitutions that are not proximal to either the Fis1 arm or the conserved surface. Thus, human Fis1 may also be autoinhibited in a manner similar to the yeast molecule. Alternatively, the Fis1 arm may mediate self-association to restrict access to the TPR-like domain, as observed in the human Fis1 crystal structure (46
In conclusion, we present data indicating that the S. cerevisiae Fis1 arm acts in an autoinhibitory manner to regulate access to a binding pocket that is evolutionarily conserved for binding the dynamin-like GTPase involved in fission of the mitochondrial outer membrane and peroxisomes (). We anticipate that this mechanism is evolutionarily conserved.