RESUMEN
Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.
Asunto(s)
Quinasas DyrK , Mitocondrias , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transducción de Señal , Proteínas Tirosina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Mitocondrias/metabolismo , Humanos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Fosforilación , Transporte de Proteínas , Células HEK293 , Células HeLa , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.
Asunto(s)
Proteínas Relacionadas con la Autofagia , Mitocondrias , Mitofagia , Fosforilación , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Humanos , Mitocondrias/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Células HeLa , Proteínas Portadoras/metabolismoRESUMEN
The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.
Asunto(s)
Mitocondrias , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fraccionamiento Celular/métodos , Mitocondrias/metabolismo , Orgánulos/metabolismo , Control de CalidadRESUMEN
Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.
Asunto(s)
Enfermedad de Alzheimer , Proteínas de Saccharomyces cerevisiae , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Drosophila melanogaster/metabolismo , Proteínas del Choque Térmico HSP40/genética , Ratones , Chaperonas Moleculares , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Membrane proteins require protein machineries to insert their hydrophobic transmembrane domains (TMDs) into the lipid bilayer. A functional analysis of protein insertases in this issue of PLOS Biology reveals that the fundamental mechanism of membrane protein insertion is universally conserved.
Asunto(s)
Membrana Dobles de Lípidos , Proteínas de la Membrana , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
A role for reversible phosphorylation in regulation of mitochondrial proteins has been neglected for a long time. Particularly, the import machineries that mediate influx of more than 1000 different precursor proteins into the organelle were considered as predominantly constitutively active entities. Only recently, a combination of advanced phosphoproteomic approaches and Phos-tag technology enabled the discovery of several phosphorylation sites at the translocase of the outer membrane TOM and the identification of cellular signalling cascades that allow dynamic adaptation of the protein influx into mitochondria upon changing cellular demands. Here, we present a protocol that allows biochemical and semi-quantitative profiling of intra-mitochondrial protein phosphorylation. We exemplify this with the pyruvate dehydrogenase complex (PDH), which serves as a central metabolic switch in energy metabolism that is based on reversible phosphorylation. Phos-tag technology allows rapid monitoring of the metabolic state via simultaneous detection of phosphorylated and non-phosphorylated species of the PDH core component Pda1. Our protocol can be applied for several further intra-organellar proteins like respiratory chain complexes or protein translocases of the inner membrane. SIGNIFICANCE: Our manuscript describes for the first time how Phos-tag technology can be applied to monitor phosphorylation of intramitochondrial proteins. We exemplify this with the regulation of the pyruvate dehydrogenase complex as central regulatory switch in energy metabolism. We show that our protocol allows a rapid monitoring of the metabolic state of the cell (phosphorylated PDH is inactive while non-phosphorylated PDH is active) and can be applied for rapid profiling of different metabolic conditions as well as for profiling phosphorylation of further intramitochondrial protein (complexes).
Asunto(s)
Mitocondrias , Proteínas Mitocondriales , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Piridinas , TecnologíaRESUMEN
Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.
Asunto(s)
Trastorno Peroxisomal , Síndrome de Zellweger , Humanos , Mitocondrias , Peroxinas/metabolismo , Trastorno Peroxisomal/metabolismo , Peroxisomas/metabolismo , Síndrome de Zellweger/metabolismoRESUMEN
The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.
Asunto(s)
Trastorno del Espectro Autista/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Trastorno del Espectro Autista/genética , Citosol/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Quinasas DyrKRESUMEN
An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Marcaje Isotópico , Espectrometría de Masas , Orgánulos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Biotina/análogos & derivados , Fraccionamiento Químico , Mitocondrias/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/metabolismo , SuccinimidasRESUMEN
The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/fisiología , Muerte Celular/fisiología , Núcleo Celular/metabolismo , ADN Mitocondrial/metabolismo , Potenciales de la Membrana/fisiología , Biosíntesis de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7-/- mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7-/- tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.
Asunto(s)
Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteína Fosfatasa 2C/genética , Proteína Fosfatasa 2C/metabolismo , Animales , Sistemas CRISPR-Cas , Clonación Molecular , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Femenino , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lipidómica , Masculino , Proteínas de Transporte de Membrana/metabolismo , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patología , Metabolómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , ProteómicaRESUMEN
The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn2+-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Mitocondrias/metabolismo , Proteínas Mitocondriales/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fosforilación , Piridinas , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , ZincRESUMEN
Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
RESUMEN
In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Mitocondrias Musculares/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/enzimología , Mitofagia/fisiología , Músculo Esquelético/enzimología , Animales , Autofagia , Quinasa de la Caseína II/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mitofagia/genética , Modelos Animales , Fosforilación , Transporte de Proteínas , Transducción de SeñalRESUMEN
Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.
Asunto(s)
ADN de Cinetoplasto/biosíntesis , ADN de Cinetoplasto/genética , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Animales , Genoma Mitocondrial , Genoma de Protozoos , Humanos , Dinámicas Mitocondriales , Membranas Mitocondriales/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/patogenicidadRESUMEN
The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are ß-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Membranas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transporte Biológico , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , NADP/metabolismo , Conformación Proteica en Hélice alfa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The mitochondrial proteome comprises ~1000 (yeast)-1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.Protein localization plays an important role in the regulation of cellular physiology. Here the authors use an integrated proteomics approach to localize proteins to the mitochondria and provide a detailed map of their specific localization within the organelle.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Humanos , Immunoblotting , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Espectrometría de Masas en TándemRESUMEN
Approximately 70% of mitochondrial precursor proteins are imported from the cytosol via N-terminal presequences, which are cleaved upon exposure to the mitochondrial processing protease MPP in the matrix. Cleaved presequence peptides then need to be efficiently degraded, and impairment of this clearance step, for example, by amyloid ß peptides, causes feedback inhibition of MPP, leading ultimately to accumulation of immature precursor proteins within mitochondria. Degradation of mitochondrial peptides is performed by Cym1 in yeast and its homologue, PreP, in humans. Here we identify the novel mitochondrial matrix protease Ste23 in yeast, a homologue of human insulin-degrading enzyme, which is required for efficient peptide degradation. Ste23 and Cym1 tightly cooperate to ensure the correct functioning of the essential presequence processing machinery.
Asunto(s)
Metaloendopeptidasas/metabolismo , Mitocondrias/metabolismo , Productos Finales de Degradación de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Humanos , Metaloproteasas/metabolismo , Mitocondrias/enzimología , Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Proteolisis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Peptidasa de Procesamiento MitocondrialRESUMEN
Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.