Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis

Abstract

Background: Extracellular 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 findings: We 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/significance: Our 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.

Figure 1. Morphology and diameter of S. cerevisiae extracellular vesicles.

A. TEM of vesicles isolated from WT (WT) and mutant cells. Each individual panel exemplifies the typical vesicle morphology for the cell group specified in the top. WT fractions shown in left panels were obtained from strain RSY113, parental of the sec4-2 mutant. Similar morphological features (not shown) were observed in vesicle fractions obtained from strain SEY6210 (parental of the snf7 mutant). Scale bar, 100 nm. B. Light scattering analysis showing diameter distribution and average values of vesicles obtained from WT (WT) or mutant (sec4-2 and snf7) cells.

Figure 2. Functional distribution of proteins in extracellular vesicle fractions obtained from S. cerevisiae WT cells.

A. Proteins were grouped by color as indicated according to their function in the cellular metabolism. B. Functional interrelationship in the collection of vesicular proteins. For protein identification according with each individual code, see Table S1.

Figure 3. Prediction of the presence of GPI-anchored sequences and signal peptide cleavage sites in S. cerevisiae cellular or vesicle proteins.

All values used in these analysis were obtained in this study, except those related to GPI-anchored sequences in cellular fractions (ψ) .

Figure 4. Venn diagrams showing similarities of protein composition in vesicle fractions from WT (RSY113 and SEY6210 strains) and mutant cells (sec4-2 and snf7 mutants).

Figure 5. Correlation analysis of the relative variation of protein abundance in vesicle fractions from WT (WT) or mutant S. cerevisiae cells.

emPAI values for each parental strain were plotted against the values obtained for yeast mutant fractions analyzed by proteomics. SEC or MVB-related mutants included snf7 (A), sec4-2 (B), vps23 (C), sec1-1 (D) and bos1-1 (E). Lower R ² values suggest greater alterations in relative protein distribution in vesicles from the mutants, in comparison to WT cells. Proteins whose abundance was increased in vesicle fractions from mutant cells are represented by the red spots, whereas proteins that were more abundant in WT fractions are highlighted in blue.

Figure 6. Sterol analysis in vesicle and cellular fractions obtained from yeast cells.

A–B. Indirect sterol-based vesicle quantification of fractions obtained from cultures of WT (WT) or mutant cells. The sterol content was determined by fluorimetric methods (A) or by densitometric analysis of bands obtained after HPTLC separation (B). Comparative analysis of the sterol content in vesicle fractions obtained from WT or mutant cells suggested that the sec4-2 mutant has a defective release of extracellular vesicles. This supposition was supported by chromatographic analysis in association with densitometry of cellular (C) or vesicle (D) fractions obtained from yeast cultures. The sec4-2 mutant, in contrast to the snf7 mutant, showed intracellular accumulation of sterols (C) and a lower kinetics of release of sterol-containing vesicles (D). Arrows indicate the migration of an ergosterol standard in TLC plates. Results are representative of three independent analyses showing similar results.

Figure 7. Sterol analysis in vesicle (A) and cellular (B) fractions obtained from WT cells and a mutant strain lacking GRASP expression.

Comparative analysis of the sterol content in vesicle fractions obtained from WT or mutant cells suggested that GRASP is involved in the release of extracellular vesicles. Chromatograms and related densitometric analyses are shown. Arrows indicate the chromatographic migration of ergosterol standards. Results are representative of three independent analyses showing similar results.