Authors & Affiliations
Zhen-Xing Huanga, Pan Zhaoa, Gui-Sheng Zenga, Yan-Ming Wanga, Ian Sudberyb, Yue Wang a,c
a Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore
b Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
c Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Published in mBio on 4 February 2014.
Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1’s cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1’s localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1-/- mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1’s function and regulation but also suggest the potential to target Nap1 in future therapeutic design.
Figure legend: Phosphorylation of Nap1 is crucial for its localization and function.
WT Nap1 (HZX02), Nap1-10A (HZX07), or Nap1-10E (HZX08) was expressed as a GFP fusion protein in nap1Δ/Δ cells. Colony morphology on GMM plates (top) and cell morphology in liquid YPD at 30°C (middle) were examined. Nap1 localization was visualized by fluorescence microscopy (bottom). (B) Co-IP of Cdc3 withWTNap1, Nap1-10A, or Nap1-10E. Cell extracts were prepared from log-phase yeast cells of HZX15 (NAP1-Myc CDC3-GFP), HZX13 (nap1-10A-Myc CDC3-GFP), and HZX14 (nap1-10E-Myc CDC3-GFP). Nap1 was immunoprecipitated with anti-Myc. Nap1 and Cdc3 in the IP products were detected with anti-Myc and anti-GFP WB, respectively. (C) The intensity of protein bands shown in panel B was determined by using ImageJ, and the ratio of Cdc3 to Nap1 in each lane was calculated. Student’s t test was performed. Error bars represent standard errors.
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