PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway

Abstract

terminal Rho1-GTP-interacting HR1 domains (Pkc1-T242) were sufficient. This indicated that the PKC1 fragments might act by interfering with Rho1-GTP signal propagation. Consistent with this, deletion of the ROM2 gene, which encodes a major Rho1p guanine-nucleotide exchange factor, bypassed the lethal effect of PtdIns(3)P accumulation in ymr1⌬ ⌬ sjl2⌬ ⌬ sjl3⌬ ⌬ triple-mutant cells. Furthermore, cells deficient in phosphoinositide 3-phosphatase (PI 3-phosphatase) activity were defective for Rho1p/Pkc1p pathway regulation, which included an inability of these cells to adapt to heat stress. Taken together, the results of this study indicated that aberrant Rho1p/Pkc1p signaling contributes to the lethal effects of PtdIns(3)P accumulation in cells deficient in PI 3-phosphatase activity.

A Detour for Yeast Oxysterol Binding Proteins

“…Other potential Osh protein effectors include other Sac1-domain phosphatases, such as Inp51/Sjl1p, Inp52/ Sjl2p, and Inp53/Sjl3p. Consistent with this idea, OSH7 overexpression suppresses the growth defects of a ymr1 ts inp52/sjl2⌬ inp53/sjl3⌬ triple phosphatase mutant (62).…”

Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae

“…However, it has been shown more recently that the myotubularin-related phosphatase, Ymr1, and two synaptojanin-like phosphatases, Inp52/Sjl2 and Inp53/Sjl3, are directly responsible for conversion of PtdIns [3]P back to PtdIns [457] (see 3.2 and 3.3 below). How the action of these enzymes is coupled to the process of MVB formation is not yet clear [455].…”

“…In S. cerevisiae Sac1 displays phosphatase activity against both PtdIns [3] [124,239,255,294,455]. In line with this, overexpression of either INP52/SJL2 or INP53/SJL3 restores several of the phenotypes associated with sac1 null mutations and the combination of a number of different null alleles of genes encoding polyphosphoinositide phosphatases display synthetic genetic interactions [294,[454][455][456][457]. Sac1 is a 623 residue type II membrane protein that possesses two transmembrane domains in its COOH-terminal part [296,458], whereas FIG 4 encodes an 879 residue enzyme lacking any other known sequence elements ( Figure 12) [450].…”

“…This strongly suggests that accumulation of PtdIns [3]P is toxic in S. cerevisiae, as the above mentioned effect depends on the catalytic activity of the targeted Sac1-like domain, whereas the generation of this lipid is dispensable for yeast cell growth (see section 2.1) [42,43]. In this regard, deletion of VPS30, encoding a subunit of Vps34 PtdIns 3-kinase complexes (see section 2.1 and Figure 5) [132], leads to a marked decrease in the cellular levels of PtdIns [3]P and, hence, restores growth of ymr1Δ inp52/sjl2Δ inp53/sjl3Δ triple mutants [455]. Likewise, a genetic screen designed to identify suppressors of the lethal phenotype of YMR1 depletion (SYD) in an inp52/sjl2Δ inp53/sjl3Δ background yielded four groups of proteins based on their cellular function, including complementing phosphatase domains of Ymr1, Inp52/Sjl2 and Inp53/Sjl3, the oxysterol-binding protein Osh7, regulators of vesicle transport such as Ypt1 and Vam6 and several components of the cell wall integrity MAP kinase pathway [455].…”

Identification and Structural Characterization of a Legionella Phosphoinositide Phosphatase*

“…The PCR products were digested with BamH1 and Xho1 or Sal1 restriction enzymes and inserted into a pET28a-based vector in-frame with an N-terminal His-SUMO tag. The same digested SidP fragments were inserted into pRS416-pGPD-GFP vector (25) for the expression of GFP fused SidP and its catalytically inactive mutant in yeast. Point mutations were generated by site-directed mutagenesis.…”