RESUMEN
Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane.
Asunto(s)
Proteínas Fúngicas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Péptido Hidrolasas/metabolismo , Transducción de Señal , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/genética , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Complejos Multiproteicos , Transporte de Proteínas , ProteolisisRESUMEN
Mitochondria are unique organelles that contain their own - although strongly reduced - genome, and are surrounded by two membranes. While most cellular phospholipid biosynthesis takes place in the ER, mitochondria harbor the whole spectrum of glycerophospholipids common to biological membranes. Mitochondria also contribute to overall phospholipid biosynthesis in cells by producing phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Considering these features, it is not surprising that mitochondria maintain highly active exchange of phospholipids with other cellular compartments. In this contribution we describe the transport of phospholipids between mitochondria and other organelles, and discuss recent developments in our understanding of the molecular functions of the protein complexes that mediate these processes. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.
Asunto(s)
Mitocondrias/metabolismo , Fosfolípidos/metabolismo , Animales , Transporte Biológico/fisiología , Cardiolipinas/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMEN
The 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 mdm10Δ 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.
Asunto(s)
Metabolismo de los Lípidos , Macrófagos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homeostasis , Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondria are essential eukaryotic organelles that are surrounded by two membranes. Both membranes contain a variety of different integral membrane proteins. After three decades of research on mitochondrial biogenesis five major import complexes with more than 40 subunits altogether were identified and characterized. In the current contribution we want to draw attention to some unexplored issues regarding the integration of mitochondrial membrane proteins and to formulate crucial questions that remain unanswered. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Asunto(s)
Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Humanos , Metabolismo de los Lípidos , Transporte de Proteínas , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Síndrome de Wolf-Hirschhorn/genética , Síndrome de Wolf-Hirschhorn/metabolismo , Proteínas de Unión al Calcio/análisis , Supervivencia Celular , Fibroblastos/citología , Eliminación de Gen , Humanos , Proteínas de la Membrana/análisis , Membranas Mitocondriales/química , Necrosis , Forma de los OrgánulosRESUMEN
The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.
Asunto(s)
ADN Mitocondrial/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Epistasis Genética , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/genética , Complejos Multiproteicos , Fenotipo , Filogenia , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Candida albicans/genética , GTP Fosfohidrolasas/metabolismo , Genes Fúngicos , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Mitochondria are unique organelles harboring two distinct membranes, the mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both mitochondrial membranes surround the hydrophilic intermembrane space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the intermembrane space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.
Asunto(s)
Retículo Endoplásmico/metabolismo , Lipasa/metabolismo , Metabolismo de los Lípidos , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Epistasis Genética , Lipasa/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Fosfatidiletanolaminas/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Aims: The outer mitochondrial membrane protein Miro1 is a crucial player in mitochondrial dynamics and calcium homeostasis. Recent evidence indicated that Miro1 mediates calcium-induced mitochondrial shape transition, which is a prerequisite for the initiation of mitophagy. Moreover, altered Miro1 protein levels have emerged as a shared feature of monogenic and sporadic Parkinson's disease (PD), but, so far, no disease-associated variants in RHOT1 have been identified. Here, we aim to explore the genetic and functional contribution of RHOT1 mutations to PD in patient-derived cellular models. Results: For the first time, we describe heterozygous RHOT1 mutations in two PD patients (het c.815G>A; het c.1348C>T) and identified mitochondrial phenotypes with reduced mitochondrial mass in patient fibroblasts. Both mutations led to decreased endoplasmic reticulum-mitochondrial contact sites and calcium dyshomeostasis. As a consequence, energy metabolism was impaired, which in turn caused increased mitophagy. Innovation and Conclusion: Our study provides functional evidence that ROTH1 is a genetic risk factor for PD, further implicating Miro1 in calcium homeostasis and mitochondrial quality control.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Anciano , Humanos , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Mutación , Proteínas de Unión al GTP rho/genéticaRESUMEN
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.
Asunto(s)
Genes Fúngicos , Mitocondrias/genética , Mitocondrias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Membrane contact sites between endoplasmic reticulum (ER) and mitochondria, mediated by the ER-mitochondria encounter structure (ERMES) complex, are critical for mitochondrial homeostasis and cell growth. Defects in ERMES can, however, be bypassed by point mutations in the endosomal protein Vps13 or by overexpression of the mitochondrial protein Mcp1. How this bypass operates remains unclear. Here we show that the mitochondrial outer membrane protein Mcp1 functions in the same pathway as Vps13 by recruiting it to mitochondria and promoting its association to vacuole-mitochondria contacts. Our findings support a model in which Mcp1 and Vps13 work as functional effectors of vacuole-mitochondria contact sites, while tethering is mediated by other factors, including Vps39. Tethered and functionally active vacuole-mitochondria interfaces then compensate for the loss of ERMES-mediated ER-mitochondria contact sites.
Asunto(s)
Retículo Endoplásmico/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Mutación Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/genéticaRESUMEN
Most mitochondrial proteins are synthesized in the cytosol prior to their import into the organelle. It is commonly accepted that cytosolic factors are required for delivering precursor proteins to the mitochondrial surface and for keeping newly synthesized proteins in an import-competent conformation. However, the identity of such factors and their defined contribution to the import process are mostly unknown. Using a presequence-containing model protein and a site-directed photo-crosslinking approach in yeast cells we identified the cytosolic chaperones Hsp70 (Ssa1) and Hsp90 (Hsp82) as well as their cochaperones, Sti1 and Ydj1, as putative cytosolic factors involved in mitochondrial protein import. Deletion of STI1 caused both alterations in mitochondrial morphology and lower steady-state levels of a subset of mitochondrial proteins. In addition, double deletion of STI1 with the mitochondrial import factors, MIM1 or TOM20, showed a synthetic growth phenotype indicating a genetic interaction of STI1 with these genes. Moreover, recombinant cytosolic domains of the import receptors Tom20 and Tom70 were able to bind in vitro Sti1 and other cytosolic factors. In summary, our observations point to a, direct or indirect, role of Sti1 for mitochondrial functionality.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Biogénesis de Organelos , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Reactivos de Enlaces Cruzados , Citosol/metabolismo , Eliminación de Gen , Genes Fúngicos , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/ultraestructura , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondria are highly dynamic organelles in all eukaryotic cells. Most of our insights regarding the mechanisms that determine the morphogenesis and motility of mitochondria have been identified and analyzed first in the model organism Saccharomyces cerevisiae. To this end high-resolution microscopic methods were applied that rely on fluorescence labeling of the organelle. A comprehensive overview of fluorescence staining approaches that were successfully applied to study the behavior of mitochondria in vivo but also in fixed cells is provided. Slightly modified versions of the methods described here can also be used to analyze other compartments of the yeast cell. Microscopic setups and imaging methods will only be shortly discussed since these are highly dependent on each laboratory's basic infrastructure.
Asunto(s)
Mitocondrias/metabolismo , Biología Molecular/métodos , Saccharomyces cerevisiae/ultraestructura , Fluorescencia , Mitocondrias/ultraestructura , Saccharomyces cerevisiae/metabolismo , Coloración y EtiquetadoRESUMEN
Genomic and proteomic studies have identified hundreds of proteins from mitochondria. A recent study has added a functional twist to these systematic approaches and identified novel mitochondrial modifiers and regulators.