AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans

Abstract

The human fungal pathogen Candida albicans is known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex. IMPORTANCE Candida albicans is a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans, C. albicans can adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2 levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition in Candida.

Essential role of the endocytic site-associated protein Ecm25 in stress-induced cell elongation

“…There are at least two possible scenarios, both of which involve control of polarized exocytosis by polarized endocytosis, a common but poorly understood problem in all eukaryotes ( Battey et al, 1999 ; Gao et al, 2003 ; Grebnev et al, 2017 ; Gundelfinger et al, 2003 ; Johansen et al, 2016 ; Jose et al, 2013 ; Marco et al, 2007 ; Wu et al, 2014 ). First, experimental and modeling studies indicate that polarized endocytosis could locally recycle polarity regulators such as Cdc42 and Rho1 as well as exocytic components such as vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (v-SNARE) to enable sustained polarized exocytosis to drive cell elongation in budding yeast ( Ayscough et al, 1999 ; Irazoqui et al, 2005 ; Jose et al, 2013 ; Marco et al, 2007 ; Orlando et al, 2011 ) as well as in filamentous fungi ( Caballero-Lima et al, 2013 ; Hernández-González et al, 2018 ; Hervás-Aguilar and Peñalva, 2010 ; Knafler et al, 2019 ; Shaw et al, 2011 ). In the absence of polarized endocytosis, such as in HU-treated ecm25Δ cells, exocytic components and polarity regulators diffuse on the bud membrane, leading to isotropic growth of the bud and a round cell morphology.…”

“…Because exocytosis and endocytosis are coordinated spatiotemporally in all eukaryotic cells ( Battey et al, 1999 ; Gao et al, 2003 ; Grebnev et al, 2017 ; Gundelfinger et al, 2003 ; Johansen et al, 2016 ; Jose et al, 2013 ; Marco et al, 2007 ; Wu et al, 2014 ), it is possible that endocytosis could play an active role in cell shape determination by affecting exocytosis. Indeed, experimental and modeling studies suggest that localized endocytosis can modulate cell shape by recycling polarity regulators and exocytic components to sites of polarized cell growth ( Caballero-Lima et al, 2013 ; Jose et al, 2013 ; Knafler et al, 2019 ; Marco et al, 2007 ; Shaw et al, 2011 ).…”

Phosphate Starvation by Energy Metabolism Disturbance in Candida albicansvip1Δ/Δ Induces Lipid Droplet Accumulation and Cell Membrane Damage

“…C. albicans wild-type strain is BWP17 [ 32 ]. For details about gene knockout strain construction or plasmid construction, please refer to our previous article [ 11 , 33 , 34 ].…”

Study on the Function of the Inositol Polyphosphate Kinases Kcs1 and Vip1 of Candida albicans in Energy Metabolism

“…The primers used to construct strains and plasmids in this study are listed in Supplementary Table S1 ; all C. albicans strains used in this study are listed in Supplementary Table S2 . C. albicans WT strain is BWP17 ( Zhang et al, 2018 ), and the gene deletion or tagging was performed on these strains as previously described ( Yu et al, 2014a ; Knafler et al, 2019 ). Briefly, the gene knockout was performed by homologous recombination, the transformed using lithium acetate and the strains were plated on SC agar medium (2% glucose, 0.2% amino acid mixture, 0.67% yeast nitrogen base, 2% agar), and the clone was selected by selective medium and confirmed by PCR.…”