RESUMEN
Lysosomes have an important role in cellular protein and organelle quality control, metabolism, and signaling. On the surface of lysosomes, the PIKfyve/Fab1 complex generates phosphatidylinositol 3,5-bisphosphate, PI-3,5-P2, which is critical for lysosomal membrane homeostasis during acute osmotic stress and for lysosomal signaling. Here, we identify the inverted BAR protein Ivy1 as an inhibitor of the Fab1 complex with a direct influence on PI-3,5-P2 levels and vacuole homeostasis. Ivy1 requires Ypt7 binding for its function, binds PI-3,5-P2, and interacts with the Fab1 kinase. Colocalization of Ivy1 and Fab1 is lost during osmotic stress. In agreement with Ivy1's role as a Fab1 regulator, its overexpression blocks Fab1 activity during osmotic shock and vacuole fragmentation. Conversely, loss of Ivy1, or lateral relocalization of Ivy1 on vacuoles away from Fab1, results in vacuole fragmentation and poor growth. Our data suggest that Ivy1 modulates Fab1-mediated PI-3,5-P2 synthesis during membrane stress and may allow adjustment of the vacuole membrane environment.
Asunto(s)
Proteínas Portadoras/metabolismo , Membranas Intracelulares/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas Portadoras/genética , Lisosomas/genética , Lisosomas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show that Ivy1, the yeast homolog of mammalian missing-in-metastasis (MIM), is a vacuolar effector of Ypt7-GTP and interacts with the EGO/ragulator complex, an activator of the target of rapamycin kinase complex 1 (TORC1) on vacuoles. Loss of Ivy1 does not affect EGO vacuolar localization and function. In combination with the deletion of individual subunits of the V-ATPase, however, we observed reduced TORC1 activity and massive enlargement of the vacuole surface. Consistent with this, Ivy1 localizes to invaginations at the vacuole surface and on liposomes in a phosphoinositide- and Ypt7-GTP-controlled manner, which suggests a role in microautophagy. Our data, thus, reveal that Ivy1 is a novel regulator of vacuole membrane homeostasis with connections to TORC1 signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Homeostasis , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Autofagia , Endocitosis , Diana Mecanicista del Complejo 1 de la Rapamicina , Modelos Biológicos , Complejos Multiproteicos , Fosfatidilinositoles/metabolismo , Unión Proteica , Saccharomyces cerevisiae/ultraestructura , Transducción de Señal , Serina-Treonina Quinasas TOR , Vacuolas/ultraestructuraRESUMEN
The endolysosomal system controls the trafficking of proteins between the plasma membrane and the degradative environment of the lysosome. The early endosomal Rab5 and the late endosomal Rab7 GTPases have a key role in the transport along the endocytic pathway by recruiting tethering factors such as the hexameric CORVET and HOPS complexes that promote membrane fusion. Both Rabs are also involved in signaling at endosomal membranes and linked to amino acid sensing and autophagy, indicating that their role in trafficking may be connected to signal transduction and adaptation during cell stress. Here, we will summarize the current knowledge on the role of both Rab GTPases on both processes and discuss the possible crosstalk between them.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo , Membrana Celular/química , Humanos , Proteínas de Unión al GTP rab/químicaRESUMEN
BACKGROUND: Netherton syndrome (NS) is a genodermatosis caused by loss-of-function mutations in SPINK5, resulting in aberrant LEKTI expression. METHOD: Next-generation sequencing of SPINK5 (NM_001127698.1) was carried out and functional studies were performed by immunofluorescence microscopy of a lesional skin biopsy using anti-LEKTI antibodies. RESULTS: We describe a novel SPINK5 likely pathogenic donor splice site variant (NM_001127698.1:c.2015+5G>A) in a patient with NS and confirm its functional significance by demonstrating complete loss of LEKTI expression in lesional skin by immunofluorescence analysis. CONCLUSION: The 2015+5G>A is a novel, likely pathogenic variant in NS. Herein we review and assimilate documented SPINK5 pathogenic variants and discuss possible genotype-phenotype associations in NS.