Sugar Responsive Ubiquitylation mediated Endosomal Trafficking of N‐acetylglucosamine Transporter (Ngt1) in Candida albicans.

Abstract

N‐acetylglucosamine (GlcNAc) signals a wide array of functions in diverse organisms ranging from bacteria to humans but in Candida albicans the dynamics associated with GlcNAc import, a prerogatory step in GlcNAc signalling is unrevealed. In this study, we have shown the molecular mechanism involved in the dynamics of a novel GlcNAc transporter (Ngt1) at the cell surface. Ngt1 showed sugar responsive endosomal trafficking to modulate its cell surface levels by interacting with key players of ubiquitylation machinery, viz. CaRsp5 ubiquitin ligase, CaArt1 adaptor protein and Snf1 protein kinase by following trafficking route of endosomes‐TransGolgi and finally vacuole. We have employed time‐lapse microscopy, LC‐MS/MS analysis, Fat‐blot technique and Site Directed Mutagenesis (SDM) along Immuno‐precipitation and immunoblot analysis to understand the intricate molecular events involved in the Endosomal trafficking. Ngt1 showed interaction with novel sphingolipids, disialoganglioside (GD3) and sulfatides that confirms its localization within lipid rafts of the plasma membrane. This study also elucidates the role of UBI4 locus and Ras1 signalling component in Ngt1 ubiquitylation and its endocytosis. Being a first of its kind report, elaborating the molecular mechanism of endosomal trafficking in Candida albicans, this study can undoubtedly provide a platform for basic research in trafficking as well as applied research towards the development of therapeutics.Support or Funding InformationScience and Engineering Research Board (SERB), Govt of IndiaGlcNAc transporter (Ngt1) undergoes ubiquitylation and internalization in response to GlcNAc.A‐Western blotting analysis showing expression, ubiquitylation and degradation of Ngt1‐GFPγ. The upshifted bands indicate ubiquitylated (Ubi) form of Ngt1‐GFPγ, supported by western blot analysis with anti‐ubiquitin antibody in panel C. B‐Localization pattern of Ngt1‐GFPγ in response to GlcNAc at 60 and 120 min after induction and it is co‐localized with a vacuole specific maker‐CMAC.Figure 1Life Cycle of Ngt1.Ngt1 is a membrane resident protein having 12 membrane‐spanning domains as predicted by TMHMM server v. 2.0. Ngt1 undergoes sugar‐responsive ubiquitylation followed by Endosomal trafficking to undergo degradation in the vacuole. The transporter shows association with novel components of the lipid rafts.Figure 2