The Mitochondrial Fission Adaptors Caf4 and Mdv1 Are Not Functionally Equivalent

<div><p>Mitochondrial fission in eukaryotes is mediated by protein complexes that encircle and divide mitochondrial tubules. In budding yeast, fission requires the membrane-anchored protein Fis1 and the dynamin-related GTPase Dnm1. Dnm1 is recruited to mitochondria via interactions with the adaptor proteins Caf4 and Mdv1, which bind directly to Fis1. Unlike Mdv1, a function for Caf4 in mitochondrial membrane scission has not been established. In this study, we demonstrate that Caf4 is a bona fide fission adaptor that assembles at sites of mitochondrial division. We also show that fission complexes may contain Caf4 alone or both Caf4 and Mdv1 without compromising fission function. Although there is a correspondence between Caf4 and Mdv1 expression levels and their contribution to fission, the two adaptor proteins are not equivalent. Rather, our functional and phylogenetic analyses indicate that Caf4 mitochondrial fission activity has diverged from that of Mdv1.</p> </div

Mitochondrial puncta containing both Caf4 and Mdv1 are fission competent.

<p>(<b>A</b>) Large panel at the left shows a representative image of <i>caf4</i>Δ <i>mdv1</i>Δ cells expressing GFP-Caf4 and RFP-Mdv1. Scale bar: 5 μm. a-f marks puncta enlarged from the image in (A) at the left. Examples of partially or completely co-localized (a–c) versus isolated (d–f) puncta are shown. Scale bar: 0.2 μm. (<b>B</b>) Quantification of Caf4 and Mdv1 colocalization. GFP- or RFP-tagged Caf4 and Mdv1 were expressed in <i>caf4Δ mdv1Δ</i> cells in the pair-wise combinations indicated. The number of GFP puncta colocalized with RFP puncta was quantified and normalized to the total number of RFP puncta in each cell (n = 10 cells). Bars and error bars are the mean and SD of three independent experiments. (<b>C</b>) Time lapse imaging of a mitochondrial fission event at a site where Caf4 and Mdv1 co-localize. Cerulean-tagged Caf4 (shown in cyan pseudo-color) and EYFP-Mdv1 were integrated at the <i>HO</i> and <i>MDV1</i> loci, respectively. Mitochondria were visualized using plasmid-borne mt-mCherry. The overlay contains true color images of all three channels. White areas indicate regions where signals from all three fluorescent proteins overlap. Scale Bar: 0.6 μm.</p

Caf4 functions independently as a mitochondrial fission adaptor.

<p>(<b>A</b>) Domain structure of the Caf4 fission adaptor, including the N-terminal extension (NTE), predicted coiled-coil (CC), and WD40 repeats predicted to form a β-propeller (WD40 repeats/ β-propeller). (<b>B</b>) Quantification of mitochondrial morphology in the indicated strains (n = 100 cells). Bars and error bars are the mean and SD of three independent experiments. (<b>C</b>) Time-lapse imaging of a mitochondrial fission event mediated by GFP-Caf4 expressed in a <i>caf4Δ mdv1Δ</i> strain. Mitochondria are labeled with mt-RFP. Scale bar: 5 µm.</p

Caf4 and Mdv1 are not functionally equivalent.

<p>(<b>A</b>) Quantification of mitochondrial morphology in a <i>caf4</i>Δ <i>mdv1</i>Δ strain expressing Caf4 or Mdv1 from the repressible <i>MET25</i> promoter in media containing different methionine concentrations (n = 100 cells). (<b>B</b>) Protein abundance of Caf4 or Mdv1 expressed from the repressible <i>MET25</i> promoter in difference methionine concentrations. The abundance of each protein was subsequently normalized to its abundance in medium containing 2.0 mM methionine. Bars and error bars are the mean and SD of three independent experiments.</p

Phylogenetic relationship of Caf4 and Mdv1 in representative fungi.

<p>The amino acid sequences of paralogs from fully sequenced fungal genomes were aligned using ClustalW2 and a phylogenetic tree was constructed using the maximum-likelihood method. Bootstrap values above 50 are shown at the nodes of the branches. Branch lengths are proportional to the number of amino acid substitutions per site. The Caf4 and Mdv1 clades are marked by vertical lines. Scale bar: 0.1 substitution per site in the protein.</p

Reanalysis of some of the Texas Aerosol data by Brodie et al [<b>3</b>].

<p>Observed and expected distribution of correlation coefficients for pairwise comparisons of intensity of detected OTUs within the classes Actinobacteria, Bacilli, Clostridia, Alpha-, Beta- and Gamma- Proteobacteria and the expected distribution of independent probe sets (black).</p

Results of the ISPMA analyses for the 64 OTU ‘sample’.

<p>The number of OTUs representing each family is proportional to the font size in this “Wordle” image. The families shown ‘pre-analyses’ (top) are represented by one (smaller text), or two (larger text) OTUs. After analyses, using a 95% detection threshold, however, some families are ‘increased’ in their OTU numbers by an order of magnitude. Families shown in red in the input sample are not present in the output set, families in green font in the output set are not present in the input sample. The inset table also shows the outputs at 90 and 92% detection thresholds.</p

Two examples of probe sharing between OTUs.

<p>On the left are shown the 56 probes that represent the OTU 6350. Probes shared with OTU 6368 are shown in the red shape, while those shared with OTU 6366 are shown in blue. The intersection of the red and blue shapes shows probes shared by all three OTUs (centre circle). All 14 and 17 probes (100% of the probe sets) for 6368 and 6366, respectively, can be found in the probe set for OTU 6350. On the right is illustrated how the complete 22 probe, probe set for OTU 5451 can be assembled from probes representing other OTUs.</p