The understanding of the processes underlying organellar function and inheritance requires the identification and characterization of the molecular components involved. We pursued a genomic approach to define the complements of genes required for respiratory growth and inheritance of mitochondria with normal morphology in yeast. With the systematic screening of a deletion mutant library covering the nonessential genes of Saccharomyces cerevisiae the numbers of genes known to be required for respiratory function and establishment of wild-type-like mitochondrial structure have been more than doubled. In addition to the identification of novel components, the systematic screen revealed unprecedented mitochondrial phenotypes that have never been observed by conventional screens. These data provide a comprehensive picture of the cellular processes and molecular components required for mitochondrial function and structure in a simple eukaryotic cell.
Wolf-Hirschhorn syndrome (WHS) is a complex congenital syndrome caused by a monoallelic deletion of the short arm of chromosome 4. Seizures in WHS have been associated with deletion of LETM1 gene. LETM1 encodes for the human homologue of yeast Mdm38p, a mitochondria-shaping protein of unclear function. Here we show that human LETM1 is located in the inner membrane, exposed to the matrix and oligomerized in higher molecular weight complexes of unknown composition. Down-regulation of LETM1 did not disrupt these complexes, but led to DRP1-independent fragmentation of the mitochondrial network. Fragmentation was not associated with changes in the levels of respiratory chain complexes, or with obvious or latent mitochondrial dysfunction, but was recovered by nigericin, which catalyzes the electroneutral exchange of K+ against H+. Down-regulation of LETM1 caused 'necrosis-like' death, without activation of caspases and not inhibited by overexpression of Bcl-2. Primary fibroblasts from a WHS patient displayed reduced LETM1 mRNA and protein, but mitochondrial morphology was surprisingly unaffected, raising the question of whether and how WHS patients counteract the consequences of monoallelic deletion of LETM1. LETM1 highlights the relationship between mitochondrial ion homeostasis, integrity of the mitochondrial network and cell viability.
Tail-anchored proteins form a distinct class of membrane proteins that are found in all intracellular membranes exposed to the cytosol. These proteins have a single membrane insertion sequence at their C-terminus and display a large N-terminal portion to the cytosol. Despite their importance for various cellular processes, the mechanisms by which these proteins are recognized at and inserted into their corresponding target membrane remained largely unclear. Here we address this issue and investigate the biogenesis of tail-anchored proteins residing in the mitochondrial outer membrane. To that goal we developed a highly specific assay to monitor the membrane insertion of the model tail-anchored protein Fis1. Using this assay, we show that in contrast to all other import pathways in yeast mitochondria, none of the import components at the outer membrane is involved in the insertion process of Fis1. Both the steady-state levels of Fis1 and its in vitro insertion into isolated mitochondria were unaffected when mitochondria mutated in known import factors were analyzed. Fis1 was inserted into lipid vesicles, and importantly, elevated ergosterol contents in these vesicles inhibited this insertion. Collectively, these results suggest that Fis1 is inserted into mitochondria in a novel pathway where the unique lipid composition of the mitochondrial outer membrane contributes to the selectivity of the process. Thus, this work demonstrates a novel role for lipids in the biogenesis of mitochondrial protein.
SummaryThe yeast mitochondrial outer membrane (MOM) protein Mdm10 is involved in at least three different processes: (1) association of mitochondria with the endoplasmic reticulum and mitochondrial lipid homeostasis (2) membrane assembly of MOM proteins, and (3) inheritance and morphogenesis of mitochondria. To decipher the precise role of Mdm10 in mitochondrial function, we screened for high-copy suppressors of the severe growth defect of the mdm10D mutant. We identified two novel mitochondrial proteins (open reading frames YOR228c and YLR253w) that we named Mdm10 complementing protein (Mcp) 1 and Mcp2. Overexpression of Mcp1 or Mcp2 restores the alterations in morphology and stability of respiratory chain complexes of mitochondria devoid of Mdm10, but the observed defect in assembly of MOM proteins is not rescued. Lipid analysis demonstrates that elevated levels of Mcp1 and Mcp2 restore the alterations in mitochondrial phospholipid and ergosterol homeostasis in cells lacking Mdm10. Collectively, this work identifies two novel proteins that play a role in mitochondrial lipid homeostasis and describes a role of Mdm10 in ergosterol trafficking.
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