Mutational Analysis of Candida albicans SNF7 Reveals Genetically Separable Rim101 and ESCRT Functions and Demonstrates Divergence in bro1-Domain Protein Interactions

Abstract

The opportunistic pathogen Candida albicans can grow over a wide pH range, which is associated with its ability to colonize and infect distinct host niches. C. albicans growth in neutral-alkaline environments requires proteolytic activation of the transcription factor Rim101. Rim101 activation requires Snf7, a member of the endosomal sorting complex required for transport (ESCRT) pathway. We hypothesized that Snf7 has distinct functions in the Rim101 and ESCRT pathways, which we tested by alanine-scanning mutagenesis. While some snf7 alleles conferred no defects, we identified alleles with solely ESCRTdependent, solely Rim101-dependent, or both Rim101-and ESCRT-dependent defects. Thus, Snf7 function in these two pathways is at least partially separable. Both Rim101-and ESCRT-dependent functions require Snf7 recruitment to the endosomal membrane and alleles that disrupted both pathways were found to localize normally, suggesting a downstream defect. Most alleles that conferred solely Rim101-dependent defects were still able to process Rim101 normally under steady-state conditions. However, these same strains did display a kinetic defect in Rim101 processing. Several alleles with solely Rim101-dependent defects mapped to the C-terminal end of Snf7. Further analyses suggested that these mutations disrupted interactions with bro-domain proteins, Rim20 and Bro1, in overlapping but slightly divergent Snf7 domains.

The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis

“…4). This extends but is consistent with previously reported data from S. cerevi- (11,17,47,49). However, we noted that all of these mutants, excepting the mvb12⌬/⌬ strain, grew more poorly than did the rim101⌬/⌬ mutant on YPD pH 9, suggesting that these ESCRT pathway members have Rim101-dependent and -independent functions during alkaline growth.…”

“…Previous studies from our lab analyzed the effects of a variety of snf7 alleles on C. albicans-stimulated epithelial cell damage (47). These studies suggested that the only contribution of Snf7 to epithelial cell damage is Rim101 dependent.…”

“…In addition to forming MVBs, the ESCRT-III member Snf7 plays a direct role in signal transduction in C. albicans and other fungi (24,47,49). Snf7 interacts with Rim20 (9,20), a scaffold protein that binds the Rim101 transcription factor (48), and with Rim13 (8,20), the putative protease responsible for Rim101 proteolytic activation ( Fig.…”

The β-Arrestin-Like Protein Rim8 Is Hyperphosphorylated and Complexes with Rim21 and Rim101 To Promote Adaptation to Neutral-Alkaline pH

“…We also found that the snf7⌬/⌬ mutant failed to repress RIM8 (Fig. 7, lanes 15 and 16), as expected, since this mutant does not activate Rim101 (28,55). However, we found that the vps4⌬/⌬ mutant also failed to repress RIM8 (Fig.…”

“…Rim101 activation requires the ESCRT-I, ESCRT-II, and ES-CRT-IIIa components of the ESCRT pathway (28,55,56,58). Thus, we asked if ESCRT components are required for Rim8 modification.…”