RESUMEN
The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III/genética , Proteínas F-Box/genética , Genes del Tipo Sexual de los Hongos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Ciclo Celular/genética , Endosomas/genética , Proteínas F-Box/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Morfogénesis/genética , Feromonas/genética , Feromonas/metabolismo , Saccharomyces cerevisiae/fisiología , Eliminación de Secuencia/genética , Transducción de Señal , Transcripción Genética , Vacuolas/genética , Vacuolas/metabolismo , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Cells typically exist in a highly dynamic environment, which cannot easily be recreated in culture dishes or microwell plates. Microfluidic devices can provide precise control of the time, dose, and orientation of a stimulus, while simultaneously capturing quantitative single-cell data. The approach is particularly powerful when combined with the genetically tractable yeast model organism. The GPCR pathway in yeast is structurally conserved and functionally interchangeable with those in humans. We describe the implementation of a microfluidic device to investigate morphological and transcriptional responses of yeast to a gradient or pulse administration of a GPCR ligand, the peptide mating pheromone α-factor.