A Rab Escort Protein Regulates the MAPK Pathway That Controls Filamentous Growth in Yeast

Abstract

1MAPK pathways regulate different responses yet can share a subset of common components. In 2 this study, a genome-wide screen was performed to identify genes that, when overexpressed, 3 induce a growth reporter (FUS1-HIS3) that responds to ERK-type MAPK pathways 4 (Mating/filamentous growth or fMAPK) but not p38-type MAPK pathways (HOG) in yeast. 5Approximately 4,500 plasmids overexpressing individual yeast genes were introduced into 6 strains containing the FUS1-HIS3 reporter by high-throughput transformation. Candidate genes 7 were identified by measuring the degree of growth, which was a reflection of reporter activity. 8Of fourteen genes identified and validated by re-testing, two were metabolic controls (HIS3 and 9 ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, 10 MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, 11 KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR 12 pathway that plays an established role in nutrient signaling. MRS6 overexpression stimulated 13 filamentous/invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. 14 Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the 15 p38/HOG pathway MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 16 by two-hybrid analysis. Collectively, the data indicate that Mrs6 may function to selectively 17 propagate an ERK-dependent signal. Generally speaking, the identification of new MAPK 18 pathway regulators by genetic screening in yeast may be a useful resource for understanding 19 signaling pathway regulation. 20 21 PC7386 MATa trpl-901PC7444 MATa ura3-52 leu2 ssk1 pRS316 [d] This study PC7445 MATa ura3-52 leu2 ssk1 pGAL-BMH1 [e] This study PC7446 MATa ura3-52 leu2 ssk1 pGAL-BMH2 [e] This study PC7447 MATa ura3-52 leu2 ssk1 pGAL-MRS6 [e] This study PC7448 MATa ura3-52 leu2 ssk1 pGAL-KAR2 [e] This study [a] Strains are in the ∑1287b background unless otherwise indicated.