Prior to this study, the Caf4 adaptor was known to function early in fission to recruit Dnm1 to the outer mitochondrial membrane. However, whether Caf4 participated in mitochondrial membrane fission after Dnm1 recruitment was unclear. Our results provide a direct demonstration that, in the absence or presence of Mdv1, Caf4 localizes in complexes on mitochondria that carry out membrane division.
It has been suggested that Caf4 serves as regulator of mitochondrial fission. We think it is unlikely that Caf4 acts as a positive regulator, since its presence or absence has little effect on Mdv1-mediated fission in WT cells. Several observations also suggest that Caf4 does not act as a negative regulator. When Caf4 is expressed in the absence of Mdv1, fission occurs and the time course of fission is similar to WT. In addition, when Caf4 and Mdv1 are both present in mitochondrial puncta, fission appears to occur normally. However, Caf4 and Mdv1 are not functionally equivalent. When expressed at maximum levels from the MET25 promoter, Caf4 (but not Mdv1) causes dominant-negative fission defects. Moreover, expression of the CAF4 gene from the MDV1 promoter in the genome is not sufficient to restore mitochondrial fission to levels observed in an MDV1 caf4Δ strain.
In many instances, one member of a duplicated gene pair is retained during evolution because it develops a specialized function (subfunctionalization) within a cellular process or because it acquires an entirely new function (neofunctionalization) 
. There is evidence that Caf4 has acquired a specialized function in mitochondrial biology. Jakob and colleagues showed previously that Caf4 (but not Mdv1) plays a role in orienting a subset of mitochondrial Dnm1 puncta toward the yeast cell cortex 
. This Dnm1 orientation is proposed to anchor mitochondria near the plasma membrane and distribute them at the cell periphery. Caf4 has also been shown to participate with Dnm1, Mdv1 and Fis1 in peroxisome division 
. Although it is possible that Caf4 has acquired specialized functions in peroxisome division, we do not observe significant changes in peroxisome number or morphology in caf4
Δ cells relative to WT (Figure S1B and C
). Moreover, cells lacking Caf4 grow as well as WT on carbon sources that require peroxisome function (i.e. oleate, Figure S1A
). If Caf4 had acquired a new function in a critical cellular process, loss of Caf4 would be expected to affect yeast fitness. However, our analysis of caf4
Δ cells has not uncovered conditions that confer a significant fitness advantage or disadvantage relative to WT with respect to cell growth on fermentable or nonfermentable carbon sources (Figure S2
), growth in the presence of a variety of drugs (Figure S2
), sporulation (Figure S3
), or growth in a head-to-head competition assay (Figure S4
). The one exception occurred when strains were grown on rapamycin, where loss of either CAF4
had a negative effect on growth relative to WT, with an additive negative growth defect in the double mutant. It remains to be seen whether this and/or other small differences observed in these studies represent bona fide fitness advantages/disadvantages that are directly attributable to the presence or absence of Caf4.
Finally, we performed domain-swapping experiments in an attempt to identify regions of Caf4 and Mdv1 that might contribute to their functional differences (Figure S5
). When expressed from the MET25
promoter on a plasmid, Mdv1 proteins containing the NTE, CC or WD40 repeats/β-propeller domains from Caf4 rescued mitochondrial morphology defects in a caf4
Δ strain as well as the WT Mdv1 protein. By contrast, swapping individual Mdv1 domains into the Caf4 protein sequence reduced fission function relative to WT Caf4. Thus, the Caf4 domains have retained fission functions that can substitute for those of Mdv1, however, there is no single domain of Mdv1 that is able to maintain or improve Caf4 function. The inability of Caf4 chimeras containing Mdv1 domains to function as well as WT Caf4 could be due to choice of domain boundaries, altered stability of the chimeric proteins and/or changes in protein structure or binding interfaces critical for fission function.
Our phylogenetic analysis revealed a slightly accelerated rate of amino acid substitution for Caf4 relative to Mdv1. However, both adaptors have retained roles in mitochondrial fission, arguing against the idea that the CAF4
gene is likely to be lost, like many other paralogs originating from whole gene duplication. Instead, Caf4 and Mdv1 appear to work synergistically, with Mdv1 carrying out the majority of fission in proportion to its higher level of expression. Consistent with this model, we find that Caf4 and Mdv1 are cross-regulated, with Caf4 expression increasing when Mdv1 is absent. The fact that Caf4 has acquired the ability to orient Dnm1 at the cell cortex 
also supports the idea that the CAF4
gene has been retained because it confers some type of selective advantage. It remains to be determined whether/how the Dnm1-orienting ability of Caf4 measurably contributes to overall fitness in yeast, and how species like S. kudriavzevii
have compensated for the loss of Mdv1.