Mitochondrial derived vesicles- Quo Vadis?

Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial-derived vesicles (MDVs) are single or double membrane-enclosed vesicles, formed and released from the mitochondrial sub-compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles.

Mitochondrial-derived vesicles retain membrane potential and contain a functional ATP synthase.

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.

Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy.

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.

Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Yeast aconitase mitochondrial import is modulated by interactions of its C and N terminal domains and Ssa1/2 (Hsp70).

Molecules of single proteins, echoforms, can be distributed between two (or more) subcellular locations, a phenomenon which we refer to as dual targeting or dual localization. The yeast aconitase gene ACO1 (778 amino acids), encodes a single translation product that is nonetheless dual localized to the cytosol and mitochondria by a reverse translocation mechanism. The solved crystal structure of aconitase isolated from porcine heart mitochondria shows that it has four domains. The first three tightly associated N-terminal domains are tethered to the larger C-terminal fourth domain (C-terminal amino acids 517-778). We have previously shown that the aconitase C terminal domain constitutes an independent dual targeting signal when fused to mitochondria-targeted passenger-proteins. We show that the aconitase N and C-terminal domains interact and that this interaction is important for efficient aconitase post translational import into mitochondria and for aconitase dual targeting (relative levels of aconitase echoforms). Our results suggest a "chaperone-like function" of the C terminal domain towards the N terminal domains which can be modulated by Ssa1/2 (cytosolic Hsp70).

Detection of Dual Targeting and Dual Function of Mitochondrial Proteins in Yeast.

Eukaryotic cells are defined by the existence of subcellular compartments and organelles. The localization of a protein to a specific subcellular compartment is one of the most fundamental processes of a living cell. It is well documented that in eukaryotic cells molecules of a single protein can be located in more than one subcellular compartment, a phenomenon termed dual targeting, bimodal targeting, or dual localization. Recently, growing evidence started to accumulate for abundant dual targeting of mitochondrial proteins, which are localized to a second location in the cell, besides this specific organelle. We have termed these dual localized proteins echoforms or echoproteins (echo in Greek denotes repetition). As the research on dual targeting of proteins is developing and evidence is accumulating for high abundance of the phenomenon, there is a growing need for new methods that would allow the identification of dual localized proteins and analysis of their functions in each subcellular compartment. This is particularly critical for single translation products that are encoded by the same gene and are actually derived from the same protein but nevertheless distribute between different subcellular compartments. The above considerations have led us to develop several approaches for studying dual localized proteins and their dual function. These include an α-complementation-based assay, specific depletion, and selection of the individual echoproteins.

A third of the yeast mitochondrial proteome is dual localized: a question of evolution.

There are a growing number of examples of identical or almost identical proteins, which are localized to two (or more) separate compartments, a phenomenon that is termed protein dual localization, dual distribution or dual targeting. We previously divided a reference set of known yeast mitochondrial proteins into two groups, suggested to be dual localized or exclusive mitochondrial proteins. Here we examined this evaluation by screening 320 mitochondrial gene products for dual targeting, using the α-complementation assay. The analysis of the results of this experimentally independent screen supports our previous evaluation that dual localized mitochondrial proteins constitute a subgroup of mitochondrial proteins with distinctive properties. These proteins are characterized by a lower probability of mitochondrial localization (MitoProtII score), a lower net charge and are enriched for proteins with a weaker mitochondrial targeting sequence. Conversely, mRNAs of exclusive mitochondrial proteins are enriched in polysomes associated with mitochondria. Based on the discovery of more than 60 new gene products that are now assumed to be dual targeted, we have updated an annotation list of dual-targeted proteins. We currently estimate that more than a third of the mitochondrial proteome is dual targeted, and suggest that this abundant dual targeting presents an evolutionary advantage.

The aconitase C-terminal domain is an independent dual targeting element.

The tricarboxylic acid cycle enzyme aconitase in yeast is a single translation product, which is dual targeted and distributed between the mitochondria and the cytosol by a unique mechanism involving reverse translocation. There is limited understanding regarding the precise mechanism of reverse translocation across the mitochondrial membranes. Here, we examined the contribution of the mature part of aconitase to its dual targeting. We created a set of aconitase mutants harboring two kinds of alterations: (1) point mutations or very small deletions in conserved sites and (2) systematic large deletions. These mutants were screened for their localization by a α-complementation assay, which revealed that the aconitase fourth domain that is at the C-terminus (amino acids 517-778) is required for aconitase distribution. Moreover, fusion of this C-terminal domain to mitochondria-targeted passenger proteins such as dihydrofolate reductase and orotidine-5'-phosphate decarboxylase, conferred dual localization on them. These results indicate that the aconitase C-terminal domain is both necessary and sufficient for dual targeting, thereby functioning as an "independent signal". In addition, the same C-terminal domain was shown to be necessary for aconitase efficient posttranslational import into mitochondria.