The small GTPase Arf1 modulates mitochondrial morphology and function.

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.

Fusion, fission, and transport control asymmetric inheritance of mitochondria and protein aggregates.

Partitioning of cell organelles and cytoplasmic components determines the fate of daughter cells upon asymmetric division. We studied the role of mitochondria in this process using budding yeast as a model. Anterograde mitochondrial transport is mediated by the myosin motor, Myo2. A genetic screen revealed an unexpected interaction of MYO2 and genes required for mitochondrial fusion. Genetic analyses, live-cell microscopy, and simulations in silico showed that fused mitochondria become critical for inheritance and transport across the bud neck in myo2 mutants. Similarly, fused mitochondria are essential for retention in the mother when bud-directed transport is enforced. Inheritance of a less than critical mitochondrial quantity causes a severe decline of replicative life span of daughter cells. Myo2-dependent mitochondrial distribution also is critical for the capture of heat stress-induced cytosolic protein aggregates and their retention in the mother cell. Together, these data suggest that coordination of mitochondrial transport, fusion, and fission is critical for asymmetric division and rejuvenation of daughter cells.

A Role of the FUZZY ONIONS LIKE Gene in Regulating Cell Death and Defense in Arabidopsis.

Programmed cell death (PCD) is critical for development and responses to environmental stimuli in many organisms. FUZZY ONIONS (FZO) proteins in yeast, flies, and mammals are known to affect mitochondrial fusion and function. Arabidopsis FZO-LIKE (FZL) was shown as a chloroplast protein that regulates chloroplast morphology and cell death. We cloned the FZL gene based on the lesion mimic phenotype conferred by an fzl mutation. Here we provide evidence to support that FZL has evolved new function different from its homologs from other organisms. We found that fzl mutants showed enhanced disease resistance to the bacterial pathogen Pseudomonas syringae and the oomycete pathogen Hyaloperonospora arabidopsidis. Besides altered chloroplast morphology and cell death, fzl showed the activation of reactive oxygen species (ROS) and autophagy pathways. FZL and the defense signaling molecule salicylic acid form a negative feedback loop in defense and cell death control. FZL did not complement the yeast strain lacking the FZO1 gene. Together these data suggest that the Arabidopsis FZL gene is a negative regulator of cell death and disease resistance, possibly through regulating ROS and autophagy pathways in the chloroplast.

Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast.

Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed genetic and proteomic interactions of Mdm33 with prohibitins, Phb1 and Phb2, which are key components of mitochondrial inner membrane homeostasis. Lipid profiling by mass spectrometry of mitochondria isolated from Mdm33-overexpressing cells revealed that high levels of Mdm33 affect the levels of phosphatidylethanolamine and cardiolipin, the two key inner membrane phospholipids. Furthermore, we show that cells lacking Mdm33 show strongly decreased mitochondrial fission activity indicating that Mdm33 is critical for mitochondrial membrane dynamics. Our data suggest that MDM33 functionally interacts with components important for inner membrane homeostasis and thereby supports mitochondrial division.

Mitochondrial ER contacts are crucial for mitophagy in yeast.

Damaged and superfluous mitochondria are removed from the cell by selective autophagy, a process termed mitophagy. This serves to maintain the proper quantity and quality of the organelle. Mitophagy is executed by an evolutionarily conserved pathway, many components of which were first discovered and characterized in yeast. In a systematic screen of a yeast deletion collection, we identified ERMES, a complex connecting mitochondria and the endoplasmic reticulum (ER), as an important factor contributing to the selective degradation of mitochondria. We show that efficient mitophagy depends on mitochondrial ER tethering. ERMES colocalizes with sites of mitophagosome biogenesis and affects the formation of the isolation membrane that engulfs the organelles destined for degradation. These results provide insights into the cellular mechanisms that govern organelle homeostasis.

ER-mitochondria contacts as sites of mitophagosome formation.

Mitophagy is a degradative process that adapts the quantity and quality of mitochondria to the cellular needs. Mitochondria destined for degradation are marked by specific receptors that recruit the core autophagic machinery to the organellar surface. The organelle is then enclosed by a phagophore (PG) which fuses with the lysosome or vacuole where the mitochondrion is degraded. In spite of significant progress in recent years, several parts of the molecular machinery of mitophagy remain unknown. We used yeast as a model organism to screen for novel components and identified the mitochondria-ER tether ERMES (ER-mitochondria encounter structure) as a major player contributing to mitophagy and formation of mitophagosomes. Tethering of mitochondria to the ER appears to be important to supply the growing PG with lipids synthesized in the ER.

Making connections: interorganelle contacts orchestrate mitochondrial behavior.

Mitochondria are highly dynamic organelles. During their life cycle they frequently fuse and divide, and damaged mitochondria are removed by autophagic degradation. These processes serve to maintain mitochondrial function and ensure optimal energy supply for the cell. It has recently become clear that this complex mitochondrial behavior is governed to a large extent by interactions with other organelles. In this review, we describe mitochondrial contacts with the endoplasmic reticulum (ER), plasma membrane, and peroxisomes. In particular, we highlight how mitochondrial fission, distribution, inheritance, and turnover are orchestrated by interorganellar contacts in yeast and metazoa. These interactions are pivotal for the integration of the dynamic mitochondrial network into the architecture of eukaryotic cells.

Analyzing membrane dynamics with live cell fluorescence microscopy with a focus on yeast mitochondria.

With the availability of increasing numbers of fluorescent protein variants and state-of-the-art imaging techniques, live cell microscopy has become a standard procedure in modern cell biology. Fluorescent markers are used to visualize the dynamic processes that take place in living cells, including the behavior of membrane-bound organelles. Here, we provide two examples of how we analyze the membrane dynamics of mitochondria in living yeast cells using wide field and confocal microscopy: (1) Long-term observation of mitochondrial shape changes using mitochondria-targeted fluorescent proteins and (2) monitoring the behavior of individual mitochondria using a mitochondria-targeted version of a photoconvertible fluorescent protein.