A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1–MPK1 cell integrity pathway

Abstract

Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1-MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-beta-glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZEO1 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP-GTP exchange factor Rom2p, while exacerbating the growth defect of sac7delta mutants at 37 degrees C. In contrast, mid2delta mutants have opposing effects to zeo1delta mutants, being synthetically lethal with rom2delta, and suppressing an 18 degrees C growth defect of sac7delta, while overexpression of MID2 rescues a rom2delta 37 degrees C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1-MPK1 cell integrity pathway.

Together we are strong—cell wall integrity sensors in yeasts

“…[5] [13], could play a vital role in signal integration. On a broader scale, CWI signalling presents an ideal target for the development of antifungal agents, which includes targeting its sensors [15].…”

“…Evidence for Wsc1 signalling directly to the GEF Rom2 within the cell was first provided by yeast two-hybrid data [39], and later shown to be inhibited by phosphorylation of serine residues in the cytoplasmic tail of Wsc1 [54]. For Mid2, interaction of its cytoplasmic tail with Rom2 seems to be further regulated by another mediator, Zeo1 [13].…”

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

“…Phosphorylation is likely to play a critical role in the communication of mitochondria with the nucleus because Ato2, Ato3, and Yat1, three proteins with a role in retrograde signaling of mitochondria (6), were found to be phosphorylated. Interestingly Pil1 and Zeo1, which are involved in the Pkc1-MAP kinase pathway (68,69), are also phosphorylated. The Pkc1-MAP kinase pathway regulates cell wall integrity and cell proliferation (68,69,73), raising the question of how this pathway should be related to mito- S62E , Atp20 S62A , or the empty vector (Ϫ) were lysed in digitonin buffer and subjected to blue native electrophoresis.…”

“…For Ato2, Ato3, and Yat1 of the lipid metabolism group, a role in mitochondrial retrograde signaling was reported (6). For Pil1 and Zeo1 a role in the Pkc1-MAP kinase pathway has been proposed (68,69). Further groups of phosphoproteins include proteins involved in redox processes, protein folding, and genome maintenance (Fig.…”

Characterization of Ccw12p, a major key player in cell wall stability of Saccharomyces cerevisiae

“…Deletion of CCW12 results in a strong hypersensitivity of the cells towards the cell wall destabilizing agents Calcofluor white and Congo red, 2.5-fold reduction of growth rate and increase in cell wall chitin (Mrsa et al, 1999;Hagen et al, 2004). CCW12 was identified as being synthetic lethal in combination with MID2, a component of the PKC1 pathway (Green et al, 2003), and with CHS3, CHS4, CHS7 and BNI4, which are involved in chitin synthesis (Lesage et al, 2005). Moreover, although ccw12 has a reduction in mating efficiency, no change in the amount of a-or α-agglutinin in the cell walls of the corresponding mating types could be seen, thus suggesting a major rearrangement of the cell wall (Mrsa et al, 1999).…”