S1–S13 Figs.

<div><p><i>Cryptococcus neoformans</i> is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. Fatal outcome of cryptococcosis depends on the ability of <i>C</i>. <i>neoformans</i> to sense and adapt to 37°C. A complex of conserved filament forming GTPases, called septins, composed of Cdc3, Cdc10, Cdc11, and Cdc12, assembles at the mother-bud neck in <i>C</i>. <i>neoformans</i>. Septins Cdc3 and Cdc12 are essential for proliferation of <i>C</i>. <i>neoformans</i> at 37°C and for virulence in the <i>Galleria mellonella</i> model of infection, presumably due to their requirement for septin complex formation, and the involvement in cytokinesis. However, how exactly Cdc3, and Cdc12 contribute to <i>C</i>. <i>neoformans</i> growth at 37°C remains unknown. Based on studies investigating roles of septins in <i>Saccharomyces cerevisiae</i>, septin complex at the mother-bud neck of <i>C</i>. <i>neoformans</i> is predicted to interact with proteins involved in cell cycle control, morphogenesis, and cytokinesis, but the septin-associated proteome in <i>C</i>. <i>neoformans</i> has not been investigated. Here, we utilized tandem mass spectrometry to define <i>C</i>. <i>neoformans</i> proteins that associate with either Cdc3 or Cdc10 at ∼25°C or after the shift to 37°C. Our findings unveil a diverse array of septin-associated proteins, highlighting potential roles of septins in cell division, and stress response. Two proteins, identified as associated with both Cdc3 and Cdc10, the actin-binding protein profilin, which was detected at both temperatures, and ATP-binding multi-drug transporter Afr1, which was detected exclusively at 37°C, were further confirmed by co-immunoprecipitation. We also confirmed that association of Cdc3 with Afr1 was enhanced at 37°C. Upon shift to 37°C, septins Cdc3 and Cdc10 exhibited altered localization and Cdc3 partially co-localized with Afr1. In addition, we also investigated changes to levels of individual <i>C</i>. <i>neoformans</i> proteins upon shift from ∼25 to 37°C in exponentially grown culture and when cells entered stationary phase at ∼25°C. Our study reveals changes to <i>C</i>. <i>neoformans</i> proteome associated with heat and nutrient deprivation stresses and provides a landscape of septin-associated <i>C</i>. <i>neoformans</i> proteome, which will facilitate elucidating the biology of septins and mechanisms of stress response in this fungal pathogen.</p></div>