Abstract
BackgroundExtracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.Methodology/Principal FindingsWe characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Conclusions/SignificanceOur results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.
Highlights
Protein secretion is a complex process that involves many organelles and accessory molecules
Fungal extracellular vesicles are believed to function as carriers of distinct molecules to the extracellular space which, in the case of pathogens, includes a wide range of virulence factors, including polysaccharides, pigments, and lipids [8,10,11,13,14]
Extracellular vesicles isolated from the yeast pathogen C. neoformans were recently reported to modulate macrophage functions [42]
Summary
Protein secretion is a complex process that involves many organelles and accessory molecules. The vesicular post-Golgi network is the most prominent example of conventional mechanism of protein secretion These proteins are sorted in the trans-Golgi network into transport vesicles that immediately move to and fuse with the plasma membrane, releasing their contents by exocytosis [1]. There are multiple unconventional mechanisms of protein secretion in eukaryotes [2] One of these mechanisms requires the formation of the exosomes, which are vesicles derived from membrane invagination within endocytic compartments (endosomes). Extracellular vesicle formation could require other cellular pathways, as suggested for Dictyostelium discoideum. In this organism, it has been hypothesized that the Golgi reassembly stacking protein (GRASP), which is attached peripherally to the cytoplasmic surface of Golgi membranes, is required for the vesicular release of acyl-CoA binding protein [4]. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown
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