Tpd3‐Pph21 phosphatase plays a direct role in Sep7 dephosphorylation in Candida albicans

Abstract

Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.

Blocking β‐1,6‐glucan synthesis by deleting KRE6 and SKN1 attenuates the virulence of Candida albicans

“…Western blotting was conducted as described by Liu et al . with minor changes (Liu et al ., ). Briefly, a cell pellet was washed and suspended in cold RIPA lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% NP‐40, 1% sodium deoxycholate) containing protease inhibitor mix and phosphatase inhibitor mix (Roche).…”

“…RNA isolation was performed largely as described previously by Liu et al . (). Cells were suspended in Trizol (Invitrogen) and broken using Tomy Microsmash and acid‐washed glass beads by five rounds of bead‐beating at 5000 rpm for 60 s with 2 min cooling on ice after each beating.…”

Candida albicans Cdc15 is essential for mitotic exit and cytokinesis

“…Indeed the initial analysis of PP1 and PP2A components in C . albicans has provided some evidence of them playing an essential role in late cell cycle progression 55 , 56 .

Figure 7 Key roles of MEN components in S .

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Candida albicans Ubiquitin and Heat Shock Factor-Type Transcriptional Factors Are Involved in 2-Dodecenoic Acid-Mediated Inhibition of Hyphal Growth

“…The transformants were verified by GFP fluorescence signal detection or Western blotting. To tag SFL1 and SFL2 at the C terminus with Myc or HA, the C terminal region of SFL1 and SFL2 was amplified, and cloned into the CIp10 vector containing either Myc or HA tag [30]. The resulting plasmid was then linearized by digestion with an enzyme located within the gene fragment and transformed into the recipient strain.…”