The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.     

Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole

Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.     

Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6.

SNARE proteins are required for fusion of transport vesicles with target membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole. Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in vti1-2 cells. Ykt6p was known to function in retrograde traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity. To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q. SNARE complexes to which Ykt6p contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function.     

The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion

The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17-independent function of Sec18. Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.     

Probing protein palmitoylation at the yeast vacuole

A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles.     

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex.     

Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding

The evolutionarily conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. While most SNAREs are permanently anchored to membranes by their transmembrane domains, the vesicle-associated SNARE Ykt6 has been found both in soluble and in membrane-bound pools. The R-SNARE Ykt6 is thought to mediate interactions between various Q-SNAREs by a reversible membrane-targeting cycle. Membrane attachment of Ykt6 is achieved by its C-terminal prenylation and palmitoylation motif succeeding the SNARE motif. In this study, we have analyzed full-length farnesylated Ykt6 from yeast and humans by biochemical and structural means. In vitro farnesylation of the C-terminal CAAX box of recombinant full-length Ykt6 resulted in stabilization of the native protein and a more compactly folded structure, as shown by size exclusion chromatography and limited proteolysis. Circular dichroism spectroscopy indicated a specific increase in the helical content of the farnesylated Ykt6 compared to the nonlipidated form or the single-longin domain, which correlated with a marked increase in stability as observed by heat denaturation experiments. Although highly soluble, farnesylated Ykt6 is capable of lipid membrane binding independent of the membrane charge, as shown by surface plasmon resonance. The crystal structure of the N-terminal longin domain of yeast Ykt6 (1-140) was determined at 2.5 Å resolution. As similarly found in a previous NMR structure, the Ykt6 longin domain contains a hydrophobic patch at its surface that may accommodate the lipid moiety. In the crystal structure, this hydrophobic surface is buried in a crystallographic homomeric dimer interface. Together, these observations support a previously suggested closed conformation of cytosolic Ykt6, where the C-terminal farnesyl moiety folds onto a hydrophobic groove in the N-terminal longin domain.     

Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.     

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the H_abc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 H_abc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the H_abc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the H_abc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the H_abc domain. In agreement, membranes carrying Vam3ΔH_abc fuse poorly unless an excess of HOPS is provided. These data suggest that the H_abc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.     

SNAREs in neurons – beyond synaptic vesicle exocytosis (Review)

The paradigm for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) function in mammalian cells has been built on advancements in our understanding of structural and biochemical aspects of synaptic vesicle exocytosis, involving specifically synaptobrevin, syntaxin 1 and SNAP25. Interestingly, a good number of SNAREs which are not directly involved in neurotransmitter exocytosis, are either brain-enriched or have distinct neuron-specific functions. Syntaxins 12/13 regulates glutamate receptor recycling via its interaction with neuron-enriched endosomal protein of 21 kDa (NEEP21). TI-VAMP/VAMP7 is essential for neuronal morphogenesis and mediates the vesicular transport processes underlying neurite outgrowth. Ykt6 is highly enriched in the cerebral cortex and hippocampus and is targeted to a novel compartment in neurons. Syntaxin 16 has a moderate expression level in many tissues, but is rather enriched in the brain. Here, we review and discuss the neuron-specific physiology and possible pathology of these and other (such as SNAP-29 and Vti1a-β) members of the SNARE family.     

Characterization of the sequence and expression of a Ykt6 prenylated SNARE from rat

The Ykt6 protein represents a novel soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE), as it is the only one known without a hydrophobic transmembrane region at the carboxy terminus. For this SNARE, however, membrane interaction is thought to be mediated through a cysteine/aliphatic/aliphatic/methionine or histidine (CAAX) C-terminal motif, a consensus sequence involved in prenylated membrane anchoring. To date, two full-length Ykt6 cDNAs have been reported, these being in yeast and human, with a further protein predicted from a Caenorhabditis elegans cosmid. Using a mouse EST clone identified as having 65% homology with the human Ykt6, we isolated a cDNA clone encoding the rat Ykt6 homolog (rYkt6). Sequence analysis of rYkt6 demonstrated that a high level of species conservation exists between the rat and human prenylated SNAREs, as both the nucleotide and amino acid sequences share >90% homology. Mammalian Ykt6 is shown here for the first time to be constitutively expressed in a variety of tissues. The species conservation and ubiquitous expression of prenylated SNAREs hence may be indicative of an important and central role for these proteins in cellular protein trafficking.     

SNAREs in neurons--beyond synaptic vesicle exocytosis (Review)

The paradigm for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) function in mammalian cells has been built on advancements in our understanding of structural and biochemical aspects of synaptic vesicle exocytosis, involving specifically synaptobrevin, syntaxin 1 and SNAP25. Interestingly, a good number of SNAREs which are not directly involved in neurotransmitter exocytosis, are either brain-enriched or have distinct neuron-specific functions. Syntaxins 12/13 regulates glutamate receptor recycling via its interaction with neuron-enriched endosomal protein of 21 kDa (NEEP21). TI-VAMP/VAMP7 is essential for neuronal morphogenesis and mediates the vesicular transport processes underlying neurite outgrowth. Ykt6 is highly enriched in the cerebral cortex and hippocampus and is targeted to a novel compartment in neurons. Syntaxin 16 has a moderate expression level in many tissues, but is rather enriched in the brain. Here, we review and discuss the neuron-specific physiology and possible pathology of these and other (such as SNAP-29 and Vti1a-beta) members of the SNARE family.     

Functional analysis of conserved structural elements in yeast syntaxin Vam3p

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.     

Ca2+ and N-ethylmaleimide-sensitive factor differentially regulate disassembly of SNARE complexes on early endosomes

The endosome-associated protein Hrs inhibits the homotypic fusion of early endosomes. A helical region of Hrs containing a Q-SNARE motif mediates this effect as well as its endosomal membrane association via SNAP-25, an endosomal receptor for Hrs. Hrs inhibits formation of an early endosomal SNARE complex by displacing VAMP-2 from the complex, suggesting a mechanism by which Hrs inhibits early endosome fusion. We examined the regulation of endosomal SNARE complexes to probe how Hrs may function as a negative regulator. We show that although NSF dissociates the VAMP-2.SNAP-25.syntaxin 13 complex, it has no effect on the Hrs-containing complex. Whereas Ca(2+) dissociates the Hrs-containing complex but not the VAMP-2-containing SNARE complex. This is the first demonstration of differential regulation of R/Q-SNARE and all Q-SNARE-containing SNARE complexes. Ca(2+) also reverses the Hrs-induced inhibition of early endosome fusion in a tetanus toxin-sensitive manner and removes Hrs from early endosomal membranes. Moreover, Hrs inhibition of endosome fusion and its endosomal localization are sensitive to bafilomycin, implying a role for luminal Ca(2+). Thus, Hrs may bind a SNARE protein on early endosomal membranes negatively regulating trans-SNARE pairing and endosomal fusion. The release of Ca(2+) from the endosome lumen dissociates Hrs, allowing a VAMP-2-containing complex to form enabling fusion.     

The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes

Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.     

The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes

The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.     

Identification of functionally interacting SNAREs by using complementary substitutions in the conserved '0' layer

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes form bundles of four parallel alpha-helices. The central '0' layer of interacting amino acid side chains is highly conserved and contains one arginine and three glutamines, leading to the classification of SNAREs into R, Qa, Qb, and Qc-SNAREs. Replacing one of the glutamines with arginine in the yeast exocytotic SNARE complex is either lethal or causes a conditional growth defect that is compensated by replacing the R-SNARE arginine with glutamine. Using the yeast SNARE complex mediating traffic from the endoplasmic reticulum to the Golgi apparatus, we now show that functionally interacting SNAREs can be mapped by systematically exchanging glutamines and arginines in the '0' layer. The Q-->R replacement in the Qb-SNARE Bos1p has the strongest effect and can be alleviated by an Q-->R replacement in the R-SNARE Sec22p. Four Q residues in the central layer caused growth defects above 30 degrees C that were rescued by Q-->R substitutions in the Qa and Qc SNAREs Sed5p and Bet1p, respectively. The sec22(Q)/sed5(R) mutant is temperature sensitive and is rescued by a compensating R-->Q replacement in the R-SNARE Ykt6p. This rescue is attributed to the involvement of Sed5p and Ykt6p in a different SNARE complex that functions in intra-Golgi trafficking.     

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting.