Isolation of a fission yeast mutant that is sensitive to valproic acid and defective in the gene encoding Ric1, a putative component of Ypt/Rab-specific GEF for Ryh1 GTPase

Abstract

Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 (+) gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (Deltaryh1) cells and Deltaric1 cells exhibited similar phenotypes. The double knockout Deltaric1Deltaryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and Deltaric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and Deltaric1 cells. Furthermore, we isolated vps15 (+) gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of Deltaric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.

Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast

“…Ryh1 regulates the intra-Golgi trafficking and is involved in recycling from endosome to the Golgi, as well as in trafficking events from the Golgi to the plasma membrane232547. Here we obtained strong evidence supporting that Ryh1 controls the activity of the CIP through an additional mechanism independent of its role as an upstream regulator of TORC2 signaling (Fig.…”

“…GTP-bound Ryh1 associates with TORC2 to induce phosphorylation and activation of Gad8 during growth, and its GTPase activity is strongly reduced under glucose starvation22. Ryh1 localizes mainly to the Golgi apparatus, and also regulates membrane trafficking from endosomes to Golgi, ER, and plasma membrane232425.…”

Genome-Wide Screening for Genes Associated with FK506 Sensitivity in Fission Yeast

“…First, we assessed the growth of Δ ryh1 and Δ ric1 cell, which showed FK506 sensitivity in our previous study [31], on YES plus FK506 or YPDA plus FK506 plates. The results showed that Δ ryh1 and Δ ric1 cells completely failed to grow on YPDA plus FK506 plates, whereas the cells partially grew on YES plus FK506 plates (data not shown).…”

GTPase Networks in Membrane Traffic

“…Both Ryh1p and Ypt3p localize to the Golgi apparatus and endosome, where they regulate protein secretion and the recycling of the exocytic SNARE from endosomes to Golgi apparatus (49). The GEF for Ryh1p is presumed to be Ric1p, a component of the Ric1p/Rgp1p complex, which in budding yeast serves as a GEF for Ypt6p (50, 51). Although the GEF activity of Ric1p toward Ryh1p is unproven and it is unclear if Ric1p is an effector of Ypt3p, one could speculate that Ryh1p and Ypt3p form a Rab GEF cascade through the Ric1p/Rgp1p complex.…”