Characterization of yeast extracellular vesicles

Evidence for the participation of different pathways of cellular traffic in vesicle biogenesis

Débora L. Oliveira, Ernesto S. Nakayasu, Luna S. Joffe, Allan J. Guimarães, Tiago J P Sobreira, Joshua D. Nosanchuk, Radames J B Cordero, Susana Frases, Arturo Casadevall, Igor C. Almeida, Leonardo Nimrichter, Marcio L. Rodrigues

Research output: Contribution to journalArticle

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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 vesicleassociated 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.

Original languageEnglish (US)
Article numbere11113
JournalPLoS One
Volume5
Issue number6
DOIs
StatePublished - 2010

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traffic
Yeast
Yeasts
yeasts
Multivesicular Bodies
Secretory Pathway
Lipids
mutants
lipids
Cells
Proteomics
proteomics
cells
rab GTP-Binding Proteins
secretion
Secretory Component
exocytosis
extracellular space
Exocytosis
transport proteins

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Characterization of yeast extracellular vesicles : Evidence for the participation of different pathways of cellular traffic in vesicle biogenesis. / Oliveira, Débora L.; Nakayasu, Ernesto S.; Joffe, Luna S.; Guimarães, Allan J.; Sobreira, Tiago J P; Nosanchuk, Joshua D.; Cordero, Radames J B; Frases, Susana; Casadevall, Arturo; Almeida, Igor C.; Nimrichter, Leonardo; Rodrigues, Marcio L.

In: PLoS One, Vol. 5, No. 6, e11113, 2010.

Research output: Contribution to journalArticle

Oliveira, DL, Nakayasu, ES, Joffe, LS, Guimarães, AJ, Sobreira, TJP, Nosanchuk, JD, Cordero, RJB, Frases, S, Casadevall, A, Almeida, IC, Nimrichter, L & Rodrigues, ML 2010, 'Characterization of yeast extracellular vesicles: Evidence for the participation of different pathways of cellular traffic in vesicle biogenesis', PLoS One, vol. 5, no. 6, e11113. https://doi.org/10.1371/journal.pone.0011113
Oliveira, Débora L. ; Nakayasu, Ernesto S. ; Joffe, Luna S. ; Guimarães, Allan J. ; Sobreira, Tiago J P ; Nosanchuk, Joshua D. ; Cordero, Radames J B ; Frases, Susana ; Casadevall, Arturo ; Almeida, Igor C. ; Nimrichter, Leonardo ; Rodrigues, Marcio L. / Characterization of yeast extracellular vesicles : Evidence for the participation of different pathways of cellular traffic in vesicle biogenesis. In: PLoS One. 2010 ; Vol. 5, No. 6.
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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 vesicleassociated 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.",
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T2 - Evidence for the participation of different pathways of cellular traffic in vesicle biogenesis

AU - Oliveira, Débora L.

AU - Nakayasu, Ernesto S.

AU - Joffe, Luna S.

AU - Guimarães, Allan J.

AU - Sobreira, Tiago J P

AU - Nosanchuk, Joshua D.

AU - Cordero, Radames J B

AU - Frases, Susana

AU - Casadevall, Arturo

AU - Almeida, Igor C.

AU - Nimrichter, Leonardo

AU - Rodrigues, Marcio L.

PY - 2010

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AB - 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 vesicleassociated 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.

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