The production of extracellular vesicles by yeast cells has now been reported for at least 6 different fungal species 
. 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 
. As described for mammalian exosomes 
, the vesicular transport in fungi may even include the release of nucleic acids to the extracellular milieu 
. The secretion of such a complex array of different molecules is expected to impact the interaction of fungal cells with their hosts. In fact, extracellular vesicles isolated from the yeast pathogen C. neoformans
were recently reported to modulate macrophage functions 
Vesicular traffic in eukaryotes is a complex and multifunctional cellular mechanism. Intracellular vesicles are required for the traffic of proteins destined for secretion, in pathways that require their movement from the endoplasmic reticulum to the Golgi complex and then, via the trans-
Golgi network, to the cell surface 
. These vesicles are expected to fuse with the plasma membrane releasing their cargo. Vesicular secretion can also involve exosomes, which originate from intracellular MVBs. These organelles derive from endosomes, which form internal vesicles that are released to the extracellular milieu as exosomes upon plasma fusion with the plasma membrane 
. Yeast extracellular vesicles have been generally termed ‘fungal exosomes’ 
, but different studies suggest that they are linked to elements of the conventional post-Golgi secretory pathway 
. It remains unknown, nevertheless, whether the formation of MVBs is related to the biogenesis of fungal extracellular vesicles.
represents the yeast cell model that extracellular vesicles have been investigated in most detail. This species appears to use trans-
cell wall vesicular transport to release its major capsular polysaccharide 
, a high molecular weight polysaccharide known as glucuronoxylomannan (GXM). Although it has been suggested that C. neoformans
produces MVB- and exosome-like structures 
, the vesicular traffic of GXM apparently requires homologues of SEC4
, suggesting that the export of polysaccharide-containing vesicles in these cells requires events of the conventional post-Golgi secretory apparatus. The events required for the biogenesis of extracellular vesicles in these cells are unclear, but the complex and variable morphology of extracellular cryptococcal vesicles analyzed by TEM 
strongly suggests that the fractions usually analyzed in studies on fungal extracellular vesicles include mixed populations of diverse cellular origin.
Although C. neoformans
was the species that led to discovery of extracellular vesicles, this fungus may not be the ideal system at this time for genetic dissection of vesicular physiology because the copious extracellular polysaccharide hinders several analytic approaches such as mass spectrometry and genetic tools remain more difficult to use relative to other model fungi. Consequently, we turned our attention to S. cerevisiae
, where we similarly detected extracellular vesicles in culture supernatants by TEM 
. Consequently we took advantage of the availability of S. cerevisiae
secretion mutants and characterized their extracellular vesicles, aiming to identify key elements required for the generation of these compartments in yeast cells. Two major prototypes were used in our study, based on previous literature observations. Snf7
, a mutant strain with defective MVB formation 
, was selected as a candidate to evaluate whether exosome formation was related to extracellular vesicles in yeast cells. The sec4-2
mutant was selected as the prototype mutant to evaluate whether events of the post-Golgi conventional secretion were required for the release of fungal extracellular vesicles, given a recent report that the orthologue of SEC4
in C. neoformans
is required for the export of polysaccharide-containing vesicles 
. Using the same rationale, some of the experiments performed in this study included mutant cells with related defects in post-Golgi secretion mechanisms (sec1-1
) and MVB biogenesis (vps23
As determined in this work and in a previous study 
, the diameter of fungal extracellular vesicles ranged from 50 to 500 nm. These dimensions contrast with the fact that extracellular vesicles in other models are in a diameter range lower than 100 nm 
. Different studies, however, have demonstrated that larger membrane structures (300–500 nm in size) are the vehicles responsible for long distance, ER-to-Golgi and trans-
Golgi to plasma membrane transport of secretory molecules (reviewed in 
). Estimation of the diameter of cell wall pores in yeast cells revealed values in the range of 200 to 400 nm 
, which would theoretically permit the release of vesicles of different sizes to the extracellular space. These observations and the high variability in the morphology of fungal extracellular vesicles 
support the hypothesis that the vesicle populations originate from compartments of distinct biogenesis, which could involve both Golgi- and exosome-derived pathways.
Proteomic analysis of yeast extracellular vesicles revealed a complex composition, as described for mammalian exosomes and other fungal vesicles 
. Several cytoplasmic proteins with no apparent relation with secretory processes were observed in the vesicle proteome, providing another parallel with mammalian exosomes. Sorting of cytosolic proteins into exosomes is normally explained by a random engulfment of small portions of cytosol during the inward budding process of MVBs 
. Of note, many cell wall-degrading enzymes were found in the S. cerevisiae
vesicles, consistent with a prior notion that the passage of vesicles through the cell wall could require hydrolysis of structural components 
. These enzymes were present in all fractions analyzed in this study. Protein composition was consistently similar in all vesicle fractions analyzed, providing confidence in the validity of the conclusion that vesicle proteins include many different functional classes.
Most of the proteins found in the yeast vesicle proteome were potentially associated other molecules identified in the vesicular protein collection. More precisely, bioinformatics analyses suggested that at least 219 protein-protein interactions were observed within the vesicle proteome. Some of these interactions were in fact expected. For example, YGR032W and YLR342W (glucan synthases) were associated, and they are in the same functional class. Apparently unrelated proteins, however, were also potentially interacting. For instance, YER103W is a heat shock protein that plays a role in protein-membrane targeting and translocation. We found that the molecule interacted with other vesicular heat shock proteins, but also with the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase 
. Similarly, YLR249W is a translational elongation factor that interacted with four other elongation factors and two heat shock proteins, but also with pyruvate decarboxylase, phosphoglucose isomerase and alcohol dehydrogenase 
. These observations illustrate the complexity of the protein composition of fungal extracellular vesicles, as well as the difficulties in unraveling their biosynthetic steps.
Vesicular fractions had greater concentrations of secretory and GPI-anchored proteins in comparison to intact S. cerevisiae cells. This observation suggests that the unconventional mechanism of protein secretion by vesicle release includes elements of the conventional secretory pathway. Furthermore, the fact that sequences potentially containing GPI anchors and signals for ER targeting are enriched in vesicle fractions confirms previous observations that fungal extracellular vesicles are actively secreted rather than released by dead cells, since they concentrate secretion-related proteins.
Our semi-quantitative analysis of protein abundance in yeast vesicles strongly suggests that fungal vesicles include compartments related to the MVB-derived pathway of exosome formation. Although vesicle release was similar in WT cells and in the snf7
mutant, the abundance of 35 proteins was modified in the mutant vesicles, suggesting that defects in MVB formation also affect extracellular vesicles in fungi. In fact, the greatest differences in protein abundance in WT/mutant systems observed in this study involved vps
mutants and, particularly, snf7
cells. In vesicle fractions from these mutants, proteins with the greatest levels of increase in relative abundance consisted of two related plasma membrane proton ATPases (YGL008C and YPL036W; Pma1p and Pma2p, respectively) and two Golgi mannosyltransferases (YDR483W and YBR199W). The mannosyltransferases YDR483W and YBR199W are known to interact with other Golgi proteins, which include, respectively, members of the exocyst complex and t-SNAREs required for vesicular transport 
. Similarly, Pma1p and its isoform Pma2p interact with several elements of Golgi-associated pathways of cellular traffic, including Ric1p, a protein involved in retrograde transport to the cis-Golgi network, and Vps29p, which is essential for endosome-to-Golgi retrograde transport 
. Therefore, we speculate that mutations in the VPS
genes could led to the activation of compensatory mechanisms of Golgi-associated traffic, which could explain the increased abundance of Golgi-related proteins in vesicles from snf7
cells. On the other hand, vesicles from the snf7
mutants had significantly decreased levels of a protein of unknown function, two glucanases and cyclophilin. The functional implication of each of these individual changes in protein abundance is unknown, but the possibilities are numerous. For instance, cyclophilin is supposed to interact with 34 different proteins in S. cerevisiae
, including elements of the secretory apparatus 
and cell wall architecture 
. These observations illustrate the fact that vesicular proteins with no apparent connections with the secretory process may be directly or indirectly linked to vesicle biogenesis.
Our results show that mutation of the SEC4
gene is associated with a delay in vesicle release to the extracellular space. This result is supportive and consistent with previous reports that a sec4-2
mutant of S. cerevisiae
and a similar mutant in C. neoformans
accumulate intracellular vesicles 
. This observation supports the view that the extracellular vesicles observed in fungal cells may not be conventional exosomes, as suggested in independent studies 
. Nevertheless, it remains unknown how post-Golgi vesicles, which are expected to fuse with the plasma membrane to release their cargo, would leave the cell wall. Different reports, however, suggest that not all secretory vesicles fuse with the plasma membrane (reviewed in 
), which supports a prior study with a sec6 C. neoformans
and our current observations with S. cerevisiae
strains showing that post-Golgi secretion events are required for the release of extracellular vesicles.
Remarkably, vesicle release was not completely abrogated in any of the mutants analyzed in this study. This observation could indicate that multiple cellular pathways are required for formation of fungal extracellular vesicles, including elements of non-conventional secretory mechanisms. In fact, in our study, mutant cells lacking expression of GRASP, which is required for unconventional secretion of an acyl coenzyme A–binding protein in S. cerevisiae 
and Dictyostelium discoideum 
, showed a decreased content of extracellular vesicles, in comparison with WT cells. This observation may be related to the fact that acyl coenzyme A–binding protein plays an important role in the cellular distribution of sphingolipids 
, which are important structural components of fungal extracellular vesicles 
. GRASP is involved in secretory mechanisms that require autophagy genes, early endosomal compartments, and MVBs 
. The decreased vesicular release by the GRASP mutant reinforces the importance of Golgi components in extracellular vesicle formation and supports the notion that extracellular release of vesicles in fungi is a multifactorial cellular event of high complexity, and possibly involves considerable redundancy. We cannot rule out the possibility, however, that the collection of mutations analyzed in our study are affecting different types of vesicles, since the methods currently used for vesicle purification do not discriminate between vesicles of different origins, resulting in heterogeneous preparations.
Our current results together with previous reports suggest agreement with the fact that endosomes and MVBs can be connected to the trans-
Golgi secretory pathway 
, which could directly affect the formation of fungal extracellular vesicles. In summary, after analysis of eight different S. cerevisiae
strains, our results indicate that both MVB- and Golgi-derived cellular pathways affect the formation and release of extracellular vesicles by fungal cells. We believe our observations with S. cerevisiae
extracellular vesicles will contribute to the understanding of a complex event in the biology of yeast cells. Since yeast extracellular vesicles in pathogens are presumably linked to fungal virulence and the ability of fungal cells to modulate the host immunity, these results could also be of use in the design of pathogenic models aiming at the elucidation of the role of secretion events in fungal virulence.