RESUMEN
Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.
Asunto(s)
Autofagosomas/metabolismo , Autofagosomas/fisiología , Fusión de Membrana/fisiología , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Animales , Eucariontes/metabolismo , Eucariontes/fisiología , Lisosomas/metabolismo , Lisosomas/fisiologíaRESUMEN
Studying the mechanism of autophagosome-vacuole fusion has proven difficult in live yeast cells. Developing a novel in vitro fusion assay, we identified Ykt6 as the missing R-SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) in this process and pinpoint the place of action of all four SNAREs involved. Parallel studies have confirmed our findings in other organisms.
RESUMEN
Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic compartment, and release the cargo for degradation. The mechanism of autophagosome-vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome-vacuole fusion. We found that this process requires the Atg14-Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome-vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process.
Asunto(s)
Autofagosomas/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fusión de Membrana , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas R-SNARE/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Vacuolas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Autophagy is a potent cellular degradation pathway, and its activation needs to be tightly controlled. Cargo receptors mediate selectivity during autophagy by bringing cargo to the scaffold protein Atg11 and, in turn, to the autophagic machinery, including the central autophagy kinase Atg1. Here we show how selective autophagy is tightly regulated in space and time to prevent aberrant Atg1 kinase activation and autophagy induction. We established an induced bypass approach (iPass) that combines genetic deletion with chemically induced dimerization to evaluate the roles of Atg13 and cargo receptors in Atg1 kinase activation and selective autophagy progression. We show that Atg1 activation does not require cargo receptors, cargo-bound Atg11, or Atg13 per se. Rather, these proteins function in two independent pathways that converge to activate Atg1 at the vacuole. This pathway architecture underlies the spatiotemporal control of Atg1 kinase activity, thereby preventing inappropriate autophagosome formation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Fagosomas/metabolismo , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
It is difficult to treat intracellular infections because of the intrinsic resistance of the microorganism to most antibiotics. Moreover, these microorganisms can survive in phagocytic cells (macrophages and neutrophils). In this study, our aims were to encapsulate an antibiotic in liposomes, which will be phagocytized as well as the microorganisms in the phagocytic cell (because liposomes were prepared using lipids which have an antigenic activity and they can be phagocytized, thus, the active substance can be transferred into the cell), and to visualise with microscopy the phagocytic activity of macrophages and neutrophils to liposomes. MLV (multilamellar vesicles) fluorescein-labeled liposomes were prepared and incubated with isolated Kangal shepherd dog macrophages and neutrophils. The phagocytosis of liposomes by monocytes was visualized step by step under the microscope. Liposomes were also observed phagocytized after incubation with neutrophils. Enrofloxacin was chosen as a model drug. Neutrophils and macrophages were isolated from Kangal shepherd dogs and infected with Staphylococcus aureus, and their phagocytic activities (PA) and microbicidal activities (MA) were determined. PA and MA values were redetermined and compared when enrofloxacin formulations were used. Liposomal enrofloxacin was found to be more effective.