Abstract

Arrestin-related trafficking (ART) proteins regulate endocytosis and turnover of nutrient transporters in fungi, yet their functional roles in Candida albicans remain largely unexplored. In this study, we identified and characterized ORF19.4887, annotated as CaECM21 in the Candida genome database, as a novel ART adaptor protein homologous to Saccharomyces cerevisiae Rod1. Sequence analysis revealed conserved PPxY motifs predicted to mediate interaction with the ubiquitin ligase Rsp5, suggesting a role in transporter endocytosis. In this study, we characterized CaECM21 by generating wild-type, mutant, and complemented strains and assessing growth under glucose, N-acetylglucosamine (GlcNAc), and amino acid starvation conditions. Transporter localization was examined through GFP fusions and fluorescence microscopy, with quantitative analyses validated by analysis of variance and post hoc testing, and amphotericin B (Amp B) sensitivity was evaluated using spot assays. Deletion of CaECM21 did not result in severe growth defects on glucose and only a moderate reduction in GlcNAc, indicating a condition-specific and modest contribution to nutrient adaptation. Our findings suggest that CaECM21 modulates nutrient transporter dynamics in response to starvation cues, potentially through ART-like PPxY motifs. Confocal imaging of GFP-tagged transporters demonstrated defective internalization of CaGAP1 in the Δ/ΔCaecm21 strain under starvation, confirming a trafficking role for CaECM21 in transporter internalization. The mutant exhibited increased sensitivity to Amp B, linking ART-mediated trafficking to membrane stress adaptation. This study reveals CaECM21 as a novel ART adaptor that contributes to nutrient-sensing and antifungal stress tolerance in C. albicans. While direct biochemical evidence for CaECM21-RSP5 remains to be established, this study provides a foundation for future mechanistic and therapeutic investigations into ART-mediated regulation of fungal physiology.

Key words: Candida albicans; CaECM21; ART adaptor; CaGAP1; Endocytosis; Amphotericin B; Nutrient transporters