RESUMEN
LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Células HeLa , Humanos , Liposomas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Mutación , Dominios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The human mitochondrion-derived calcium transporter Letm1 was synthesized by reconstituted in vitro transcription-translation (IVTT) in cell-sized liposomes and the dependency of Letm1 on phospholipid composition was investigated. Components for IVTT were encapsulated into cell-sized vesicles together with the DNA encoding Letm1, thereby preparing proteoliposomes. The synthesis of Letm1 and pH-dependent calcium transport activity were confirmed by flow cytometry. Finally, we investigated the effect of phospholipid composition on Letm1 transport activity and found that cardiolipin present in the mitochondrial membrane plays an important role on the transport activity of Letm1.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de la Membrana/genética , Fosfolípidos/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Citometría de Flujo , Humanos , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fosfolípidos/químicaRESUMEN
The coordination of subcellular processes during adaptation to environmental change is a key feature of biological systems. Starvation of essential nutrients slows cell cycling and ultimately causes G1 arrest, and nitrogen starvation delays G2/M progression. Here, we show that budding yeast cells can be efficiently returned to the G1 phase under starvation conditions in an autophagy-dependent manner. Starvation attenuates TORC1 activity, causing a G2/M delay in a Swe1-dependent checkpoint mechanism, and starvation-induced autophagy assists in the recovery from a G2/M delay by supplying amino acids required for cell growth. Persistent delay of the cell cycle by a deficiency in autophagy causes aberrant nuclear division without sufficient cell growth, leading to an increased frequency in aneuploidy after refeeding the nitrogen source. Our data establish the role of autophagy in genome stability through modulation of cell division under conditions that repress cell growth.
Asunto(s)
Autofagia/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Mitosis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Transcripción , Aminoácidos/genética , Aminoácidos/metabolismo , Aneuploidia , Proliferación Celular , Fase G1/genética , Fase G2/genética , Inestabilidad Genómica , Nitrógeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
DNA replication without telomerase leads to telomere shortening and induces replicative senescence. We found that in a telomerase-deficient budding yeast mutant, the volume of each telomere-shortened cell increased as its growth capacity decreased, and that this process was associated with changes in vacuolar morphology. Senescence-induced cell expansion required Mec1, a DNA damage-responsive kinase, but not vacuolar SNARE Vam3.