Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling

Kening Zhao,Michael Liem,Lahiru Gangoda,Ivan K Poon,Vincent Bulone,Peter Lock,Kuok Yap,Ching-Seng Ang,David Chisanga,Hina Kalra,Haidar Al Saffar,Lanzhou Jiang,Suresh Mathivanan,Pamali Fonseka,Shivakumar Keerthikumar,Mark Bleackley,Christopher G Adda,Marilyn Anderson

doi:10.1038/s42003-019-0538-8

Abstract

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.

Highlights

Extracellular vesicles (EVs) are membranous vesicles that are released by cells
EVs were isolated from wild type (WT) and Endosomal Sorting Complex Required for Transport (ESCRT) knockout strains that had been grown for 18 h before the culture medium was collected and subjected to differential centrifugation coupled with ultracentrifugation
Very few particles between 30 and 150 nm were detected in the 100k pellet from heat-killed cells (Supplementary Fig. 1b) confirming that the EVs isolated from the growing cultures were not fragments of dead cells

Summary

Introduction

The role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks[1] and chitin synthase Chs[3], in yeast EVs. Interestingly, EVs containing Fks[1] and Chs[3] rescued the yeast cells from antifungal molecules. It is well established that the endosomal sorting complex required for transport (ESCRT) machinery is critical for the biogenesis of exosomes in mammalian cells[7,8]. Though fungal EVs have been reported to contain cell wall remodelling enzymes[12], their role in cell wall remodelling has not been examined

Methods

Results

Conclusion