Doctor of Philosophy

dissertationThe mitochondrion is an essential organelle in eukaryotic cells. The major functions of mitochondria are ATP production, calcium homeostasis, heme and lipid biosynthesis and apoptosis. Mitochondria form a dynamic tubular spaghetti-like network that fuses, divides and moves within the cell. The molecular events that regulate mitochondrial dynamics are important for various cellular and developmental processes and are linked to cellular dysfunction and disease. This dissertation focuses on the molecular machineries that carry out mitochondrial fission in yeast and human. Mitochondrial fission requires both dynamin-related GTPases (called Dnm1/Drp1 in yeast/human) and membrane adaptors. In yeast, a tail-anchored protein called Fis1 and an adaptor protein called Mdv1 recruit Dnm1 to the membrane. Mdv1 interacts with Dnm1 and stimulates Dnm1 self-assembly. Although Fis1 is conserved in humans, an Mdv1 ortholog is absent. Instead, humans have at least three other membrane anchored proteins (Mff, MiD49, MiD51/MIEF1) whose roles in fission are poorly defined. Studies presented in this thesis address two key issues related to mitochondrial fission. First, experiments in Chapter 2 address how the structure of the yeast mitochondrial adaptor (Mdv1) affects Dnm1 function. A structural study of the Mdv1 central domain shows that this region forms an unusually long antiparallel coiled coil which positions the P-propeller domains of Mdv1 to interact with Dnm1 as it transitions from the cytoplasm to mitochondria. In vivo studies with altered coiled coil domains demonstrates the importance of this domain in Mdv1-Fis1 binding, Dnml mitochondrial recruitment and Dnm1-mediated mitochondrial fission. Second, experiments in Chapter 3 determine whether multiple human adaptors are capable of working independently to elicit Drp1-mediated membrane fission. In vivo studies using the yeast and human adaptors in a minimal yeast system show that Fis1 is dispensable for mitochondrial membrane scission. These studies also demonstrate that Mdv1, Mff, or MiDs can work in parallel with their corresponding DRPs to catalyze membrane fission. Importantly, coassembly of MiD49 protein with Drp1 dramatically decreases the diameter of assembled Drp1 structures. Together, these studies advance our understanding of how adaptors and fission dynamins work together to achieve membrane constriction and fission, and raise important questions for future studies

Molecular architecture of a dynamin adaptor: implications for assembly of mitochondrial fission complexes

Structural and functional studies of the Mdv1 dimer reveal how this mitochondria-associated adaptor protein orients and stabilizes the assembly of dynamins on membranes to promote mitochondrial fission

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

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

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

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 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