Calmodulin Binding to Dfi1p Promotes Invasiveness of Candida albicans

Abstract

Candida albicans, a dimorphic fungus, undergoes hyphal development in response to many different environmental cues, including growth in contact with a semi-solid matrix. C. albicans forms hyphae that invade agar when cells are embedded in or grown on the surface of agar, and the integral membrane protein Dfi1p is required for this activity. In addition, Dfi1p is required for full activation of mitogen activated protein kinase Cek1p during growth on agar. In this study, we identified a putative calmodulin binding motif in the C-terminal tail of Dfi1p. This region of Dfi1p bound to calmodulin in vitro, and mutations that affected this region affected both calmodulin binding in vitro and invasive filamentation when incorporated into the full length Dfi1p protein. Moreover, increasing intracellular calcium levels led to calcium-dependent, Dfi1p-dependent Cek1p activation. We propose that conformational changes in Dfi1p in response to environmental conditions encountered during growth allow the protein to bind calmodulin and initiate a signaling cascade that activates Cek1p.

The two transmembrane regions of Candida albicans Dfi1 contribute to its biogenesis

“…An untagged strain showed no fluorescence (Figure 4, panel A). The strain encoding full-length Dfi1-GFP showed fluorescence at the cell periphery (Figure 4, panel B), as previously described [6,7]. The strain encoding Dfi1 ΔN -GFP showed fluorescent signal at the cell periphery (Figure 4, panel C).…”

“…Here we experimentally determined that mature Dfi1 contains two transmembrane domains. This and other data [7] suggest a model of Dfi1 topology and membrane orientation where the N- and C-termini are cytoplasmic, and the inter-transmembrane region is extracellular. Because the N-terminal transmembrane domain is retained in mature Dfi1 protein, this domain and/or the intracellular N-terminal region may mediate or regulate Dfi1 signaling.…”

“…Interestingly, mutation of other functionally-important motifs within the Dfi1 C-terminus [6,7] did not cause this marked increase in Dfi1 quantity. Therefore, a yet-uncharacterized feature of the Dfi1 C-terminus may regulate Dfi1 protein levels.…”

“…Mutant DFI1 alleles were constructed by overlap PCR with mutagenic primers using pSPLDFI1-TAP [6,7] as template. Primers are listed in Table S2.…”

“…Dfi1 is an integral plasma membrane protein that undergoes both O- and N-linked glycosylation [6]. The Dfi1 C-terminus contains motifs that are important for Dfi1 function [6,7], but the role of the Dfi1 N-terminus remains relatively unexplored.…”