Loss of Num1-mediated cortical dynein anchoring negatively impacts respiratory growth.

Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to the canonical functions of dynein in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.

Cox15 interacts with the cytochrome bc1 dimer within respiratory supercomplexes as well as in the absence of cytochrome c oxidase.

The heme a molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. Heme a is synthesized from heme o by a multi-spanning inner membrane protein, heme a synthase (Cox15 in the yeast Saccharomyces cerevisiae). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. To improve our understanding of heme a insertion into cytochrome c oxidase, here we investigated the protein-protein interactions that involve Cox15 in S. cerevisiae In addition to observing Cox15 in homooligomeric complexes, we found that a portion of Cox15 also associates with the mitochondrial respiratory supercomplexes. When supercomplex formation was abolished, as in the case of stalled cytochrome bc1 or cytochrome c oxidase assembly, Cox15 maintained an interaction with select proteins from both respiratory complexes. In the case of stalled cytochrome bc1 assembly, Cox15 interacted with the late-assembling cytochrome c oxidase subunit, Cox13. When cytochrome c oxidase assembly was stalled, Cox15 unexpectedly maintained its interaction with the cytochrome bc1 protein, Cor1. Our results indicate that Cox15 and Cor1 continue to interact in the cytochrome bc1 dimer even in the absence of supercomplexes or when the supercomplexes are destabilized. These findings reveal that Cox15 not only associates with respiratory supercomplexes, but also interacts with the cytochrome bc1 dimer even in the absence of cytochrome c oxidase.

Mitochondria-ER-PM contacts regulate mitochondrial division and PI(4)P distribution.

The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.

The multifunctional nature of mitochondrial contact site proteins.

Mitochondria make physical contact with nearly every other membrane in the cell, and these contacts have a wide variety of functions that are carried out by proteins that reside at the sites of contact. Over the past decade, tremendous insight into the identity and functions of proteins localized to mitochondrial contact sites has been gained. In doing so, it has become clear that one protein or protein complex can contribute to contact site formation and function in a wide variety of ways. Thus, complex and often surprising relationships between the roles of a mitochondrial contact site and its multifunctional resident proteins continue to be unraveled.