Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway

Abstract

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 ␤␥ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by G␤␥ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein ␣ 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. Many extracellular signals are detected by G-proteincoupled receptors (GPCRs). 10 In animals, these signals include odors, tastes, light, pH, nucleotides, biogenic amines, peptides, steroids, and phospholipids. In each case, receptor activation results in binding of GTP to a G-protein ␣ subunit and dissociation of G␣ from the ␤␥ subunit dimer. Both G␣ and G␤␥ can then transduce signals through the activation of intracellular enzymes and ion channels. G-protein signaling ends when GTP is hydrolyzed to GDP, a process that is accelerated by members of the regulator of G-protein signaling (RGS) family (1). GPCRs also play an important role in yeast mating. Genetic analysis in yeast Saccharomyces cerevisiae has led to the identification and characterization of several new signaling proteins, including the first RGS protein Sst2 (2). Sst2 is required for pheromone gradient tracking (3-5), adaptation (6, 7), and noise suppression (8). Other pathway components were identified from mutants that produce an unresponsive sterile (ste) phenotype, including the mating pheromone receptors (Ste2 and Ste3), the G-protein ␤␥ subunits (Ste4 and Ste18), a G␤␥ effector (Ste5), downstream protein kinases (Ste20, Ste11, and Ste7), and a transcription factor (Ste12) (2). Further analysis revealed that the G-protein ␤␥ dimer recruits and activates Far1 in complex with Cdc24 (9-13), an exchange factor for the small G-protein Cdc42 (14), as well as Ste5, a scaffold protein required for activation of the MAPKs Fus3 and Kss1 (15-22). Cdc42 promotes cell cy...

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association

“…Another PTM that may be involved in the regulation of the pheromone signaling pathway is ubiquitination [ 75 ]. Ste18 was detected as a ubiquitinated protein in a large-scale MS analysis [ 41 ], with positions K26 and K41 identified as potential ubiquitination sites.…”

Regulation of large and small G proteins by ubiquitination

“…In a follow-up analysis, it was shown that while the F-box protein Cdc4 selectively targets Gpa1, other F-box proteins target downstream components of the pheromone-signaling pathway as follows: Dia2 selectively limits pheromone-induced vacuolar targeting; Ucc1 selectively limits MAPK induction; and Pfu1 is needed for proper mating morphogenesis and the disassembly of unproductive mating projections (45).…”

Potassium starvation induces autophagy in yeast

“…Prominent among these are the mammalian muscarinic cholinergic and βadrenergic receptors, which bind to acetylcholine and epinephrine, respectively (73-78). Likewise, we have shown that the pheromone GPCR in yeast promotes vacuolar delivery of cytoplasmic contents (38,79). Whereas the response to nitrogen and potassium requires Complex I of the yeast PI 3-kinase, mating pheromone requires Complex II.…”

“…To enable high-throughput comparison of autophagy responses across diverse nutritional conditions, we used Rosella, a fluorescent reporter of autophagy ( Figure 1A) (37,38). Rosella is comprised of super-ecliptic pHluorin (green, pH sensitive) fused with DsRed.T3 (red, pH stable).…”