Endocytosis is not essential for the endosome formation

Rab5 is a key regulator of endosome fusion and trafficking, but whether endocytotic vesicle internalization is necessary for Rab5 function has not been determined. To clarify this, we utilized two yeast mutants, sla2Δ and sac6Δ scp1Δ mutants, which have defects in clathrin-mediated endocytosis32,33, and a control strain lacking the Vps21p GEFs, Vps9p, and Muk1p, which regulate the endosomal localization of Vps21p28. We first confirmed the endocytic defect in these mutants using Alexa Fluor 594-labeled yeast mating pheromone α-factor (A594-α-factor), a marker of endocytosis34,35. Consistent with previous reports32,33, sla2Δ and sac6Δ scp1Δ mutants exhibited severe defects in the uptake of A594-α-factor from the PM, while in wild-type cells the majority of A594-α-factor was transported to the vacuole by 20 min after addition (Fig. 1a, b). In the vps9Δ muk1Δ mutant, Alexa-α-factor was internalized normally but accumulated in multiple endosomal compartments at 20 min after addition (Fig. 1a, b), suggesting a delay of α-factor transport to the vacuole after internalization. To further confirm the endocytic defect, we next examined the internalization of 3-triethylammoniumpropyl-4-p-diethylaminophenylhexatrienyl pyridinium dibromide (FM4-64), a lipophilic styryl dye that is used to follow bulk membrane. When added to wild-type cells, FM4-64 is immediately incorporated into the PM and internalized via bulk-phase endocytosis, and then transported to the vacuole within 20 min (Supplementary Fig. 1a, b). Similar to Alexa-α-factor uptake, the sac6Δ scp1Δ mutant showed a remarkable defect in the uptake of FM4-64 from the PM and the vps9Δ muk1Δ mutant showed a delay of FM4-64 movement to the vacuole after internalization (Supplementary Fig. 1a, b). Since yeast also has a clathrin-independent endocytic pathway that depends on the Rho1 GTPase36, we generated a triple mutant lacking the Rho1-GEF, Rom1p, in addition to Sac6p and Scp1p. The sac6Δ scp1Δ rom1Δ triple mutant exhibited a more severe defect in FM4-64 uptake from the PM, compared to the scp1Δ rom1Δ mutant (Supplementary Fig. 1a, b). We next examined the localization of Vps21p in these mutants. To precisely evaluate differences in the localization of Vps21p, each mutant was compared directly alongside wild-type cells that were labeled by their expression of Vph1p-mCherry (Fig. 1c). We previously demonstrated that Vps21p is predominantly localized at the EE-to-LEs, and little localized at the TGN15. Consistent with this observation, in the wild-type cell GFP-Vps21p was localized at multiple endosomal compartments, whereas deletion of the VPS9 and MUK1 genes led to the complete relocalization of Vps21p to the cytosol (Fig. 1c, d). In contrast, the sla2Δ and sac6Δ scp1Δ mutants exhibit Vps21p localization similar to that in wild-type cells (Fig. 1c). The sac6Δ scp1Δ rom1Δ triple mutants also exhibited Vps21p localization similar to that in wild-type cells (Fig. 1c). By comparing the localization of GFP-Vps21p with tdTomato-tagged Hse1p, a marker of the EE-to-LEs37,38, we confirmed that GFP-Vps21p localizes at endosomal compartments in sac6Δ scp1Δ cells, similar to wild-type cells (Supplementary Fig. 1c, d), as described previously15. Quantitative analysis revealed that the number of GFP-Vps21p-labeled endosomes in the endocytosis-defective mutants is almost the same as that in wild-type cells (Fig. 1d). The fluorescence intensity of GFP-Vps21p on endosomes was also not significantly changed in the endocytosis-defective mutants, compared to wild-type cells (Fig. 1e). Similar results were obtained when we used other endocytic mutants, such as end3Δ and myo3Δ myo5Δ (Fig. 1d, e). Since Vps21p functions in the VPS pathway from the TGN to the vacuole, as well as the endocytic pathway, we examined if the vacuolar pathway is intact in the endocytic mutants by using Vph1-GFP as a marker15,31. As expected, in the vps9Δ muk1Δ mutant that blocks the VPS pathway, Vph1-GFP accumulated in multiple puncta, similarly to what is seen in the vps21Δ mutant15; however in the sla2Δ and sac6Δ scp1Δ mutants it is normally transported to the vacuole (Fig. 1a). The number and fluorescence intensity of GFP-Vps21p-labeled endosomes was also not significantly changed in cells treated with Latrunculin A, which abolishes both clathrin-dependent and clathrin-independent endocytosis (Supplementary Fig. 1e–g)36,39. Additionally, we found that the number and fluorescence intensity of structures labeled with Vps8-GFP, which is a marker for the late or prevacuolar endosome11, are not changed in the sac6Δ scp1Δ mutants (Supplementary Fig. 1h–j). These results indicated that Vps21p is normally localized and functions at the endosome in these endocytic mutants, and thus, endocytic internalization is not essential for Vps21p-mediated endosome formation.

Fig. 1 Defective endocytosis does not affect Vps21p-mediated endosome formation. a Effect of the deletion of Rab5-specific GEFs or endocytosis-related proteins on the internalization of Alexa Fluor594-labeled α-factor (Alexa-α-factor) or Vhp1-GFP transport to the vacuole. The images were acquired at 0, 5, and 20 min after washing out unbound Alexa-α-factor (Alexa-α-factor). b Quantification of the intracellular compartments accumulating Alexa-α-factor in the indicated cells at 20 min after internalization. The compartments were categorized into four classes; plasma membrane only (PM), PM and endosome and/or vacuole (PM + end./vac.), endosome and/or vacuole (end./vac.), and vacuole only (vac.). c Localization of GFP-Vps21p in wild-type (WT) and mutant cells. WT and mutant cells expressing GFP-Vps21p were grown to early-logarithmic to mid-logarithmic phase, mixed, and acquired in the same images. Fluorescence images or heat maps showing GFP levels are shown in the panels labeled GFP-Vps21p or GFP intensity, respectively. WT or mutant cells are indicated with red or yellow dashed lines, respectively. WT cells are labeled by the expression of Vph1-mCherry (red) which is shown in the lower images overlaid with DIC images. d, e Quantification of the (d) number or (e) fluorescence intensity of GFP-Vps21p-positive endosomes displayed in (c). Data show mean ± SEM from at three independent experiments, (b) with 50 cells or (e) 100 endosomes, or (d) mean ± SD with 150 cells. *p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., not significant, chi-square test for trend (b). Different letters indicate significant difference at p < 0.0001, one-way ANOVA with Tukey’s post-hoc test (d, e). Scale bar in all panels, 2.5 μm Full size image

Post-Golgi transport is required for the endosome formation

We wished to determine if an alternative trafficking pathway could fuel endosome formation and focused on the VPS pathway from the TGN which converges with the endocytic pathway at an early stage of endocytosis, independently of yeast Rab5s15. We first examined the requirement for vesicle transport from the TGN for Vps21p activation. We utilized two major drugs, Brefeldin A (BFA) and Monensin, which perturb post-Golgi transport by inhibiting ER–Golgi or intra-Golgi traffic40,41. Intriguingly, treatment of wild-type cells with BFA or Monensin caused similar defects in Alexa-α-factor transport to those seen in the vps9Δ muk1Δ or vps21Δ mutant:15 Alexa-α-factor was internalized normally but accumulated in multiple dots, localizing GFP-Vps21p, at 20–30 min after addition (Fig. 2a, Supplementary Figs. 2a, and 3a). In BFA-untreated or Monensin-untreated cells, colocalization of Alexa-α-factor with GFP-Vps21p-labeled dots is only transient at 5–10 min after internalization but in BFA-treated or Monensin-treated cells the colocalization was still maintained after 10–30 min (Supplementary Figs. 2c and 3d), suggesting that these drug treatments might inhibit the process of endosome fusion mediated by Vps21p. By comparing the localization of GFP-Vps21p with Sec7p or Hse1p, markers for the TGN or endosomes15, respectively, we found that Vps21p is localized at endosomal compartments in BFA-treated cells, as well as in untreated cells (Supplementary Fig. 2d, e). Quantitative analysis categorizing the Alexa-α-factor localization as endosome only, endosome and vacuole, or vacuole only, revealed that these drugs inhibit the transport of Alexa-α-factor at the Vps21p-residing endosome (Fig. 2b, Supplementary Fig. 3b). We further analyzed temporal changes in the number of Alexa-α-factor-labeled endosomes. In wild-type cells, the number increased by 5 min, and then rapidly decreased until most Alexa-α-factor had been transported to the vacuole (Fig. 2c, Supplementary Fig. 3c). In contrast, the number of Alexa-α-factor-labeled endosome did not substantially change after reaching a maximum in BFA or Monensin-treated cells (Fig. 2c, Supplementary Fig. 3c). We observed a similar delay upon utilizing FM4-64 in BFA-treated or Monensin-treated cells (Fig. 2d, e, Supplementary Fig. 3e, f). Since changes in the number of A594-α-factor-labeled endosomes in cells treated with these drugs were quite similar to those in vps21Δ ypt52Δ cells15, we speculated that these drugs might affect the activation status of Vps21p. To examine this, we observed the localization of Vps21p as an indirect readout of its activity because the GTP-bound active form is targeted to the endosomal membranes and the GDP-bound inactive form is localized in the cytosol42,43 (Supplementary Fig. 4a). As expected, treatment of wild-type cells with these drugs changed the Vps21p localization from the endosome to the cytosol in a time-dependent manner (Fig. 2f and Supplementary Fig. 4b). We observed that Vps21p transiently accumulates (Fig. 2f and Supplementary Fig. 4c, 5–30 min), and then gradually disperses in the cytosol (Fig. 2f and Supplementary Fig. 4c, 10–60 min). We note that BFA treatment changed Arf1p localization within 5 min, and also induce accumulation of Vps21p with the same timing (Supplementary Fig. 4c). At 60 min (BFA) or 30 min (Monensin) after drug treatment, the intensity of GFP-Vps21p at endosomes decreased to ~37% or ~23% of that in the wild-type cells and increased in the cytosol to ~1.5 or ~1.9 fold that of the wild-type cells in the BFA-treated or Monensin-treated cells, respectively (Fig. 2g and Supplementary Fig. 4d). The intensity of Vps8-GFP at endosomes also decreased to ~27% at 30 min after Monensin treatment (Fig. 2h, i). These observations suggest that vesicle transport from the TGN is important for Vps21p-mediated endosomal transport to the vacuole.

Fig. 2 The effect of inhibiting post-Golgi traffic on Vps21p-mediated vesicle formation and trafficking. a The spatio-temporal localization of Alexa-α-factor in Brefeldin A-treated cells. Cells were labeled with Alexa-α-factor in the presence or absence of 100 μg m−1 L−1 Brefeldin A (BFA). The images were acquired at the indicated time after internalization of Alexa-α-factor. b Quantification of Alexa-α-factor localization in the cells at 20 min after internalization. Endosome only (end.), endosome and vacuole (end. + vac.) and vacuole only (vac.). c Quantification of the number of Alexa-α-factor-positive vesicles displayed in a. d Effect of BFA treatment on FM4-64 transport from the PM to the vacuole. After treatment of the cells with 100 μg m−1 L−1 BFA for 15 min, cells were labeled with 200 μM FM4-64 for 15 min on ice and observed at 0, 20, and 40 min after washing out unbound FM4-64 and incubating the cells at 25 °C. e Quantification of FM4-64 localization in the cells at 40 min after internalization. Puncta only (punc.), puncta and vacuole (punc. + vac.), and vacuole only (vac.). f The effect of BFA on the localization of Vps21p. Cells expressing GFP-Vps21p were incubated with 100 μg m−1 L−1 BFA at 25 °C and observed at the indicated time after the incubation. Red arrows indicate the example of GFP-Vps21p endosomes and yellow arrow shows aberrant accumulation of GFP-Vps21p. g The graph shows quantification of the fluorescence intensity of GFP-Vps21p at the endosomes and in the cytoplasm. h The effect of Monensin on the localization of Vps8p. Cells expressing Vps8-GFP were incubated with 50 μM Monensin at 25 °C and observed at the indicated time after the incubation. i The graph shows quantification of the fluorescence intensity of Vps8-GFP at the endosomes and in the cytoplasm. Data show mean ± SEM from three independent experiments, with 50 cells b, c, 100 cells e or 100 endosomes g, i. ****p < 0.0001, chi-square test for trend b, e, two-way ANOVA with Bonferroni’s post-hoc test c. *p < 0.05, unpaired t-test with Welch’s correction g, i. Scale bar in all panels, 2.5 μm Full size image

Arf1p and Ent3p/5p are required for Vps21p activation

We sought to identify Golgi-resident proteins that are required for the regulation of Vps21p activity on the endosome. Arf1p has been demonstrated to be a key regulator of Golgi-to-vacuole transport44. Thus, we next examined the effect of deleting the ARF1 gene on Vps21p. We first examined if GFP-Vps21p localizes at the endosome in the arf1Δ mutant by comparing the localization with Hse1-tdTomato. We found that GFP-Vps21p dots in the arf1Δ mutants colocalize with Hse1-tdTomato, similar to what is seen in wild-type cells (Supplementary Fig. 5a, b). In the arf1Δ mutant, the number of the endosomes containing GFP-Vps21p was increased (Fig. 3a–c), whereas the fluorescence intensity at the endosomes was decreased relative to that in wild-type cells (Fig. 3a, d), suggesting that Vps21p-mediated endosomal transport is impaired in this mutant. To further confirm the requirement for post-Golgi transport in endosomal transport, we utilized cells lacking the clathrin adaptor proteins that regulate transport from the TGN to the endosome. In S. cerevisiae, three classes of TGN-resident adaptors, the AP-1 complex, the Gga homolog Gga1p/2p and epsin-related Ent3p/5p, have been identified44,45. By expressing GFP-Vps21p in cells lacking single or multiple adaptors, we found that a double deletion of ENT3 and ENT5 genes causes relocalization of much of the endosomal GFP-Vps21p to the cytosol, similar to BFA-treated cells or the arf1Δ mutant (Fig. 3a–d and Supplementary Fig. 5c). GFP-Vps21p dots in the ent3Δ ent5Δ mutant also colocalized with Hse1-tdTomato (Supplementary Fig. 5a, b), indicating that these dots are endosomes. In the ent3Δ ent5Δ double mutant, the fluorescent intensity of GFP-Vps21p-labeled endosomes was significantly decreased (Fig. 3d), whereas the number of the endosomes was increased (Fig. 3c). These observations suggest a possibility that decreased endosomal localization of activated Vps21p reduces the competence of endosomal transport, causing increase of punctate compartments labeled with GFP-Vps21p in this mutant. In contrast, deletion of Apl4p, the γ-subunit of the AP-1 complex, Gga1p/2p, or even a triple deletion of these adaptors, had a negligible effect on the number and fluorescent intensity of Vps21p endosome (Supplementary Fig. 5c). These results, therefore, support the idea that Arf1p-mediated and Ent3p/Ent5p-mediated post-Golgi transport is important for Vps21p-mediated endosomal transport.

Fig. 3 The effect of deleting Arf1p or adaptor proteins on the localization and activity of Vps21p. a, b The localization of GFP-Vps21p in wild-type (WT) and mutant cells. Fluorescence images were acquired as shown in Fig. 1c. High magnification images indicated by arrowhead in a were shown in b. c Quantification of the number of GFP-Vps21p-positive endosomes displayed in a and Supplementary Fig. 2a. d Fluorescence intensity of GFP-Vps21p-positive endosomes displayed in c. e Immunoblots showing active levels of Vps21p. Endogenous Vps21p was tagged with GFP in the indicated cells, and 3 μg of total cell lysate (2% input) were loaded and immunoblotted with an anti-GFP antibody (Input panel). Active Vps21p from 150 μg of total cell lysate was pulled down with GST-tagged N terminal portion of human EEA1 (GST-EEA1NT) and probed with an anti-GFP antibody (Pulldown panel). f Quantification of active Vps21p levels displayed in e. Graph shows mean ± SEM from three independent experiments. Data show mean ± SD with 150 cells c or mean ± SEM with 100 endosomes d from three independent experiments. Different letters indicate significant difference at p < 0.05, one-way ANOVA with Tukey’s post-hoc test c, d, f. Scale bar, 2.5 μm. Uncropped blots for e can be found in Supplementary Fig. 11 Full size image

We further investigated if post-Golgi transport is required for the activity of Vps21p using a pull-down assay. The active forms of Rab5A/B have been shown to interact directly with the N-terminal region (1-209 a.a.) of human EEA1 (EEA1NT)46 (Supplementary Fig. 6a). We purified GST-fused EEA1NT (Supplementary Figs. 6a, b, 10) and confirmed that it specifically binds to the GTP-bound form of Vps21p (Vps21Q66L), but not the GDP-bound form of Vps21p (Vps21S21N) (Supplementary Fig. 6c). Using GST-EEA1NT, we next tested the amount of the active GTP-bound form of Vps21p in wild-type cells and in the mutants that affect post-Golgi transport. We first examined the functionality of the assay, and showed that Vps21p was efficiently pulled down from the cell lysate prepared from wild-type cells, but rarely from extract lacking the Vps21p-GEFs (Fig. 3e, f), indicating that GST-EEA1NT specifically binds to the active Vps21p. Consistent with the observed normal Vps21p localization at endosomes in the sac6Δ scp1Δ mutant (Fig. 1c–e), a similar amount of Vps21p was pulled down from this double mutant (Fig. 3e, f) as from the wild-type cells. In contrast, the active Vps21p was significantly decreased in arf1Δ or ent3Δ ent5Δ mutants to 37 ± 5% or 36 ± 5% of the wild-type cells, respectively (Fig. 3e, f). Furthermore, expression of the GTP-bound form of Vps21p partially suppresses the growth defect of the arf1Δ mutant at 37 °C (Supplementary Fig. 6d). Thus, Arf1p-mediated and Ent3p/5p-mediated post-Golgi transport seems to be important for the Vps21p activation.

Arf1p and Ent3/5p are required for Vps9p localization

We wondered if the importance of post-Golgi transport for Vps21p activity might be due to an effect on Vps9p. Vps9p was found at similar levels in wild-type and in arf1Δ or ent3Δ ent5Δ mutant cells (Fig. 4a) so we focused on its localization. Live-cell imaging of GFP-Vps9p revealed Vps9p at several puncta, in addition to the cytosol in wild-type cells (Fig. 4b), as reported previously28. Interestingly, we found that the number of puncta containing GFP-Vps9p (Fig. 4c) and its residence time at the puncta (Fig. 4d–f, and Supplementary Movie 1) were increased relative to wild-type in the ent3Δ ent5Δ mutant. To make Vps9p localization clearer, we expressed GFP-Vps9p under the control of the ZWF1 gene promoter, which moderately increased its expression, compared with the authentic promoter (Supplementary Figs. 7a and 10)47. We obtained similar results showing increased Vps9p puncta and increased residence time of Vps9p at the puncta in the ent3Δ ent5Δ mutant (Supplementary Fig. 7b–f). Through comparisons with Sec7p or Hse1p, we found that Vps9p is predominantly localized at the endosomes in wild-type cells, but that deletion of the ENT3 and ENT5 genes significantly increased Vps9p’s localization at the TGN and decreased it at the endosomes (Fig. 4g, h). Taken together with the observations that Vps21p is localized to the cytosol and displays a decreased activity in the ent3Δ ent5Δ mutant, these results suggest that decreased localization of Vps9p at the endosomes might affect the activity of Vps21p in the ent3Δ ent5Δ mutant.

Fig. 4 Arf1p and Ent3p/5p-dependent localization of Vps9p. a Immunoblots showing the expression levels of GFP-tagged Vps9p in the cells. GFP-Vps9p was expressed under the control of the authentic promoter from the endogenous locus. Total cell lysates were loaded and immunoblotted with an anti-GFP antibody (α-GFP panel). GAPDH was used as a loading control (α-GAPDH panel). Graph shows mean ± SEM from three independent experiments. b Localization of GFP-Vps9p in the cells. Fluorescence images (GFP-Vps9p), heat maps showing GFP levels (GFP intensity) and DIC images (DIC) are shown. c Quantification of the number of GFP-Vps9p puncta displayed in b. d, e Dynamic behavior of GFP-Vps9p puncta in the cells. Time series of the regions in the boxed area indicated in d. Blue and red arrowheads indicate appearing and disappearing points of GFP-Vps9p. f Graph shows the GFP-Vps9p lifetime in the cells. n = 100 puncta. Top and bottom bars are the 95% confidence limits. Data show the mean ± SEM of three independent experiments, with 100 cells. g Colocalization of GFP-Vps9p and Sec7p-mCherry (TGN) or Hse1p-tdTomato (Endosome; End) in the cells. Representative intensity profiles of GFP-Vps9p and Sec7-mCherry or Hse1-tdTomato along the yellow line in the merged images are indicated in the lower graphs. Yellow or red/green arrowheads indicate the presence or absence of colocalization, respectively. h The percentages of colocalization were calculated as the ratio of mCH/tdTom-tagged marker (n = 100) colocalizing with GFP-Vps9p-positive puncta. i Analysis of the interaction between Arf1p and Vps9p using the BioID assay. Vps9p and Arf1p were tagged with GFP and BirA*-S, respectively. Total cell lysate (1% input) or biotinylated proteins were loaded and immunoblotted with an anti-GFP (α-GFP panel) or anti-S-tag antibody (S-tag panel). Data show mean ± SD with 150 cells c or mean ± SEM with 100 puncta f, h from three independent experiments. Different letters indicate significant difference at p < 0.05, one-way ANOVA with Tukey’s post-hoc test a and c. ****p < 0.0001, unpaired t-test with Welch’s correction f. ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey’s post-hoc test h. Scale bar in all panels, 2.5 μm. Uncropped blots for a and i can be found in Supplementary Fig. 11 Full size image

We also wished to examine the effect of ARF1 gene deletion on Vps9p localization. Deletion of the ARF1 gene also impaired the proper localization and activation of Vps21p (Fig. 3), but we could not precisely assess the effect on Vps9p’s TGN localization because of the high fluorescence intensity in the cytosol (Fig. 4b). Previous studies demonstrated that Vps9p accumulates at aberrant endosomes deemed class E compartment in cells lacking Vps4p, which catalyzes the release of the ESCRT complex from the endosomal membrane28,30. Since in vps4Δ cell the fluorescence intensity of Vps9p in the cytosol was low enough to assess the intensity at the endosomal compartments, we examined the effect of the ARF1 gene deletion on Vps9p localization, using this mutant. We observed the accumulation of GFP-Vps9p at the prevacuolar endosomal compartment in the vps4Δ mutant, which was decreased upon the additional deletion of the ARF1 gene (Supplementary Fig. 7g, h). This suggests a role for Arf1p in Vps9p recruitment to endosomal compartments. In addition, we utilized the BioID assay to examine the interaction between Arf1p and Vps9p. We fused bacterial biotin ligase BirA (R118G) mutant (BirA*) to Arf1p and expressed this hybrid protein to be able to biotinylate endogenous proteins that interact with Arf1p. Pull-down analysis with Streptavidin-agarose demonstrated that BirA*-tagged Arf1p could biotinylate Vps9p in vivo (Fig. 4i). These results are consistent with a potential role for Arf1p in the recruitment of Vps9p to the TGN before its transport to the endosome where it catalyzes nucleotide exchange on Vps21p.

Ypt31p/32p are required for Vps9p transport to the endosome

Recent studies have reported a functional relationship between Arf1p and Ypt31p/32p (yeast Rab11) at the trans-Golgi/TGN48, thus we next examined if Ypt31p/32p play a role in Vps9p-mediated Vps21p activation. In wild-type cells, Vps9p was minorly localized at the TGN labeled by Sec7-mCherry and highly localized at endosomes labeled by Hse1-tdTomato (Fig. 5a, b). Interestingly, the TGN localization of Vps9p increased, and the endosome localization decreased in the ypt31(K127N) ypt32Δ temperature-sensitive (ypt31ts) mutant49 at the non-permissive temperature (37 °C) (Fig. 5a, b and Supplementary Fig. 8a). The localization of Vps9p in the ypt31ts mutant at 37 °C was similar to that in the ent3Δ ent5Δ mutant (Fig. 4g, h), and this motivated us to further examine the localization of Vps21p and Ent3/5p in the ypt31ts mutant. As expected, at 37 °C the number of GFP-Vps21p-positive endosomes was significantly increased (Fig. 5c–e), whereas the fluorescence intensity at the endosomes was decreased in the ypt31ts mutant, compared to that in wild-type cells (Fig. 5f). Furthermore, we observed that in the ypt31ts mutant Ent3p and Ent5p clearly change their localization to the cytosol at 37 °C, although they are normally localized at the TGN at 25 °C (Fig. 5g). Quantitative analysis revealed that the fluorescent intensities of Ent3p or Ent5p at the TGN labeled by Sec7-GFP significantly decreases at 37 °C compared to that seen there at 25 °C (Fig. 5g, h). The fluorescent intensity of mCherry-tagged Apl2p, a β-subunit of AP-1 complex, decreases at 37 °C compared to that seen there at 25 °C, but this change seems not to be significant because the intensity of Sec7-GFP also decreases in the ypt31ts mutant at 37 °C (Fig. 5h). These results suggest a requirement for Ypt31p/32p function for the recruitment of Ent3/5p to the TGN, and subsequent Vsp9p-mediated Vps21p activation.

Fig. 5 Ypt31p/32p-dependent localization of Vps9p at endosomes. a Colocalization of GFP-Vps9p and Sec7p-mCherry (Sec7-mCH) or Hse1p-tdTomato (Hse1-tdTom) in wild-type or ypt31-temperature-sensitive (ypt31ts) mutant cells. Ypt31p function was diminished by incubating cells at 37 °C for 2 h. Representative intensity profiles of GFP-Vps9p and Sec7-mCherry or Hse1-tdTomato along the yellow line in the merged images are indicated in the lower graphs. Yellow or red/green arrowheads indicate the presence or absence of colocalization, respectively. b The percentages of colocalization were calculated as the ratio of mCH/tdTom-tagged marker (n = 100) colocalizing with GFP-Vps9p-positive puncta in each experiment. c Localization of GFP-Vps21p in the cells. Fluorescence images (GFP-Vps21p), heat maps showing GFP levels (GFP intensity) are shown. WT cells are labeled by the expression of Vph1-mCherry (red) which is shown in the images overlaid with DIC images. d High magnification images of the cells indicated with white arrows in c are shown. e, f Quantification of the number e or fluorescence intensity f of GFP-Vps21p dots displayed in c. g Localization of mCherry-tagged Ent3p (ENT3-mCH), Ent5p (ENT5-mCH), or Apl2p (APL2-mCH) in ypt31ts cells. Sec7-GFP was expressed as a control to evaluate the effect of Ypt31p dysfunction on Golgi/TGN function. h Quantification of the fluorescence intensity of mCherry-fused Ent3p, Ent5p, and Apl2p at the TGN (as labeled by Sec7-GFP) in ypt31ts cells. Intensity of Sec7-GFP was used as a control. Data show mean ± SEM with 100 puncta b, f, h or mean ± SD with 150 cells e from three independent experiments. ***p < 0.001, ****p < 0.0001, n.s., not significant, two-way ANOVA with Tukey’s post-hoc test b. Different letters indicate significant difference at p < 0.05, one-way ANOVA with Tukey’s post-hoc test e and f. **p < 0.01, n.s., not significant, unpaired t-test with Welch’s correction h. Scale bar in all panels, 2.5 μm Full size image

We wished to further confirm that Ypt31p/32p are needed for Vps21p activity. We first tested if Vps21p’s cellular localization in another condition similarly requires Ypt31p/32p. Upon the deletion of the yeast Rab5-specific GAP Msb3p, Vps21p accumulates on the vacuolar membrane, due to being fixed into its GTP-bound form20,43. We observed this also in the ypt31ts mutant at 25 °C, but at 37 °C when Ypt31p function is inhibited, Vps21p accumulation was significantly diminished (Supplementary Fig. 8b, c). We then wished to test the effects of altering Ypt31p more subtly, altering its nucleotide state rather than its entire functionality. We tested Trs120p, a specific subunit of the TRAPPII complex that is reported to function as a GEF for Ypt31p50. Indeed, we found that membrane-bound Ypt31p is decreased in trs120 knockdown (kd) cells (Supplementary Fig. 8d) and that expression of a GTP-bound form of Ypt31p rescues the defective growth of trs120kd cells (Supplementary Fig. 8e). In trs120kd cells, Vps21p was partly dispersed in the cytosol, similar to what we observed in the ypt31ts mutant (Supplementary Fig. 8f). In msb3Δ cells, the trs120kd also shifted some Vps21p from the vacuolar membrane to the cytosol (Supplementary Fig. 8g). These observations support our conclusion that Ypt31p/32p can function as upstream regulators for Vps21p.

Vps9-CUE domain cooperates with Arf1p in Vps21p activation

The localization of Vps21p at endosomes was significantly decreased but not completely inhibited in the arf1Δ mutant (Fig. 3d), suggesting that other mechanisms redundantly regulate Vps9p localization. One possible mechanism is the ubiquitin-dependent localization of Vps9p through its CUE domain that binds to mono-ubiquitinated endocytic cargo51,52,53. We found that deletion of Vps9p’s CUE domain has little effect on Vps21p’s localization, but results in a remarkable decrease in the fluorescent intensity of the GFP-Vps21p dots when combined with the arf1Δ mutation (Fig. 6a, b). By directly comparing the arf1Δ mutant with the vps9ΔCUE arf1Δ double mutant, we found that the fluorescent intensity of GFP-Vps21p dots in the double mutant decreases compared to that seen in the arf1Δ mutant (Fig. 6c, d). This suggests that Vps21p activation mediated by the Vps9-CUE domain is independent of that mediated by Arf1p. The number of Vps21p-positive dots was decreased in the vps9ΔCUE arf1Δ mutant compared to that in arf1Δ cells (Fig. 6e). Pull-down analysis with GST-fused EEA1NT demonstrated that the amount of GTP-bound Vps21p does not change in the vps9ΔCUE mutant, but is significantly decreased in the vps9ΔCUE arf1Δ double mutant, although it was still higher than that seen in vps9Δ cells (Fig. 6f, g). A previous study reported that Arf2p, which is 96% identical to Arf1p but expressed ~10-fold lower than Arf1p, has a redundant function with Arf1p54, and therefore, Arf2p might partially substitute for Arf1p in the vps9ΔCUE arf1Δ mutant.