RESUMEN
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.
Asunto(s)
Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Bioensayo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Sistema Libre de Células , Ergosterol/farmacología , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Conejos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle.
Asunto(s)
Proteínas Portadoras/metabolismo , Citocromo-B(5) Reductasa/metabolismo , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Citocromo-B(5) Reductasa/análisis , Citocromo-B(5) Reductasa/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.