Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2010 Jun 19

PMID 20559436

Citations 140

Authors

Debora L Oliveira

Ernesto S Nakayasu

Luna S Joffe

Allan J Guimaraes

Tiago J P Sobreira

Joshua D Nosanchuk

Radames J B Cordero

Susana Frases

Arturo Casadevall

Igor C Almeida

Leonardo Nimrichter

Marcio L Rodrigues

Affiliations

Soon will be listed here.

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.

Citing Articles

A cell fractionation and quantitative proteomics pipeline to enable functional analyses of cotton fiber development.

Lee Y, Rani H, Mallery E, Szymanski D Plant J. 2025; 121(4):e17246.

PMID: 39970036 PMC: 11838819. DOI: 10.1111/tpj.17246.

Endocytic tethers modulate unconventional GAPDH secretion.

Cohen M, Philippe B, Lipke P Cell Surf. 2025; 13():100138.

PMID: 39830088 PMC: 11742311. DOI: 10.1016/j.tcsw.2024.100138.

Extracellular vesicles in phytopathogenic fungi.

Rutter B, Innes R Extracell Vesicles Circ Nucl Acids. 2024; 4(1):90-106.

PMID: 39698296 PMC: 11648432. DOI: 10.20517/evcna.2023.04.

Systematic characterization of extracellular vesicles from potato ( cv. Laura) roots and peels: biophysical properties and proteomic profiling.

Ekanayake G, Piibor J, Midekessa G, Godakumara K, Dissanayake K, Andronowska A Front Plant Sci. 2024; 15:1477614.

PMID: 39619839 PMC: 11607679. DOI: 10.3389/fpls.2024.1477614.

Hidden allies: how extracellular vesicles drive biofilm formation, stress adaptation, and host-immune interactions in human fungal pathogens.

Brandt P, Singha R, Ene I mBio. 2024; 15(12):e0304523.

PMID: 39555918 PMC: 11633191. DOI: 10.1128/mbio.03045-23.

References

Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, Tamura N . Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles. Endocrinology. 2007; 148(8):3850-62. DOI: 10.1210/en.2006-1479. View

Wuestehube L, Duden R, Eun A, Hamamoto S, Korn P, Ram R . New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. Genetics. 1996; 142(2):393-406. PMC: 1206974. DOI: 10.1093/genetics/142.2.393. View

Novick P, Field C, Schekman R . Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980; 21(1):205-15. DOI: 10.1016/0092-8674(80)90128-2. View

Novick P, Schekman R . Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979; 76(4):1858-62. PMC: 383491. DOI: 10.1073/pnas.76.4.1858. View

Schirle M, Heurtier M, Kuster B . Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol Cell Proteomics. 2003; 2(12):1297-305. DOI: 10.1074/mcp.M300087-MCP200. View

Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J . Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics. 2005; 4(9):1265-72. DOI: 10.1074/mcp.M500061-MCP200. View

Nickel W, Rabouille C . Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol. 2009; 10(2):148-55. DOI: 10.1038/nrm2617. View

Rodrigues M, Nimrichter L, Oliveira D, Frases S, Miranda K, Zaragoza O . Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell. 2006; 6(1):48-59. PMC: 1800364. DOI: 10.1128/EC.00318-06. View

Duran J, Anjard C, Stefan C, Loomis W, Malhotra V . Unconventional secretion of Acb1 is mediated by autophagosomes. J Cell Biol. 2010; 188(4):527-36. PMC: 2828925. DOI: 10.1083/jcb.200911154. View

10.

Nosanchuk J, Nimrichter L, Casadevall A, Rodrigues M . A role for vesicular transport of macromolecules across cell walls in fungal pathogenesis. Commun Integr Biol. 2009; 1(1):37-39. PMC: 2629580. DOI: 10.4161/cib.1.1.6639. View

11.

Albuquerque P, Nakayasu E, Rodrigues M, Frases S, Casadevall A, Zancope-Oliveira R . Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol. 2008; 10(8):1695-710. PMC: 2562661. DOI: 10.1111/j.1462-5822.2008.01160.x. View

12.

Schuldiner M, Collins S, Thompson N, Denic V, Bhamidipati A, Punna T . Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell. 2005; 123(3):507-19. DOI: 10.1016/j.cell.2005.08.031. View

13.

Walworth N, Goud B, Kabcenell A, Novick P . Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989; 8(6):1685-93. PMC: 401010. DOI: 10.1002/j.1460-2075.1989.tb03560.x. View

14.

Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear E, Sevier C . The genetic landscape of a cell. Science. 2010; 327(5964):425-31. PMC: 5600254. DOI: 10.1126/science.1180823. View

15.

Keller S, Sanderson M, Stoeck A, Altevogt P . Exosomes: from biogenesis and secretion to biological function. Immunol Lett. 2006; 107(2):102-8. DOI: 10.1016/j.imlet.2006.09.005. View

16.

Oliveira D, Nimrichter L, Miranda K, Frases S, Faull K, Casadevall A . Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan. Fungal Genet Biol. 2009; 46(12):956-63. PMC: 2778009. DOI: 10.1016/j.fgb.2009.09.001. View

17.

Mears R, Craven R, Hanrahan S, Totty N, Upton C, Young S . Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics. 2004; 4(12):4019-31. DOI: 10.1002/pmic.200400876. View

18.

Takeo K, UESAKA I, Uehira K, Nishiura M . Fine structure of Cryptococcus neoformans grown in vivo as observed by freeze-etching. J Bacteriol. 1973; 113(3):1449-54. PMC: 251716. DOI: 10.1128/jb.113.3.1449-1454.1973. View

19.

Gaigg B, Neergaard T, SCHNEITER R, Hansen J, Faergeman N, Jensen N . Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae. Mol Biol Cell. 2001; 12(4):1147-60. PMC: 32293. DOI: 10.1091/mbc.12.4.1147. View

20.

Nicola A, Frases S, Casadevall A . Lipophilic dye staining of Cryptococcus neoformans extracellular vesicles and capsule. Eukaryot Cell. 2009; 8(9):1373-80. PMC: 2747831. DOI: 10.1128/EC.00044-09. View