Dual targeting of mitochondrial proteins: Mechanism, regulation and function

AbstractOne solution found in evolution to increase the number of cellular functions, without increasing the number of genes, is distribution of single gene products to more than one cellular compartment. It is well documented that in eukaryotic cells, molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized proteins are coined in this review “echoforms” indicating repetitious forms of the same protein (echo in Greek denotes repetition) distinctly placed in the cell. This term replaces the term to “isoproteins” or “isoenzymes” which are reserved for proteins with the same activity but different amino acid sequences. Echoforms are identical or nearly identical, even though, as referred to in this review may, in some cases, surprisingly have a totally different function in the different compartments. With regard to mitochondria, our operational definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a mitochondrial membrane.In this review we ask how, when and why mitochondrial proteins are dual localized in the cell. We describe mechanisms of dual targeting of proteins between mitochondria and other compartments of the eukaryotic cell. In particular, we have paid attention to situations in which dual localization is regulated in time, location or function. In addition, we have attempted to provide a broader view concerning the phenomenon of dual localization of proteins by looking at mechanisms that are beyond our simple definition of dual targeting. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

In eukaryotes, each subcellular compartment harbors a specific group of proteins that must accomplish specific tasks. Nfs1 is a highly conserved mitochondrial cysteine desulfurase that participates in iron-sulfur cluster assembly as a sulfur donor. Previous genetic studies, in Saccharomyces cerevisiae, have suggested that this protein distributes between the mitochondria and the nucleus with biochemically undetectable amounts in the nucleus (termed “eclipsed distribution”). Here, we provide direct evidence for Nfs1 nuclear localization (in addition to mitochondria) using both α-complementation and subcellular fractionation. We also demonstrate that mitochondrial and nuclear Nfs1 are derived from a single translation product. Our data suggest that the Nfs1 distribution mechanism involves at least partial entry of the Nfs1 precursor into mitochondria, and then retrieval of a minor subpopulation (probably by reverse translocation) into the cytosol and then the nucleus. To further elucidate the mechanism of Nfs1 distribution we determined the N-terminal mitochondrial sequence of Nfs1 by Edman degradation. This led to the discovery of a novel mitochondrial processing enzyme, Icp55. This enzyme removes three amino acids from the N terminus of Nfs1 after cleavage by mitochondrial processing peptidase. Intriguingly, Icp55 protease (like its substrate Nfs1) appears to be dual distributed between the nucleus and mitochondria

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2

10.1007/s00294-017-0786-4Current Genetics643697-71

Yeast Aconitase in Two Locations and Two Metabolic Pathways: Seeing Small Amounts Is Believing

The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the α peptide of the β-galactosidase enzyme to aconitase and observed α complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution “eclipsed distribution.

Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell

Previous studies demonstrated that dysfunctional yeast proteasomes accumulate in the insoluble protein deposit (IPOD), described as the final deposition site for amyloidogenic insoluble proteins and that this compartment also mediates proteasome ubiquitination, a prerequisite for their targeted autophagy (proteaphagy). Here, we examined the solubility state of proteasomes subjected to autophagy as a result of their inactivation, or under nutrient starvation. In both cases, only soluble proteasomes could serve as a substrate to autophagy, suggesting a modified model whereby substrates for proteaphagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD. Furthermore, the insoluble fraction accumulating in the IPOD represents an alternative pathway, enabling the removal of inactive proteasomes that escaped proteaphagy when the system became saturated. Altogether, we suggest that the relocalization of proteasomes to soluble aggregates represents a general stage of proteasome recycling through autophagy

Ubiquitination Occurs in the Mitochondrial Matrix by Eclipsed Targeted Components of the Ubiquitination Machinery

Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research

Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response.

In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation