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Rab GAP cascade regulates dynamics of Ypt6 in the Golgi traffic
Associated Data
Significance
The Golgi apparatus functions as the central station of membrane traffic in cells. A series of Rab GTPases, which control various steps in membrane traffic, act consecutively during the course of Golgi maturation. Here, we report that Ypt6, a Rab6 homologue in yeast, resides temporarily at the Golgi and dissociates into the cytosol upon arrival of Ypt32, another Rab GTPase functioning in the late Golgi. We have found that Gyp6, a putative GTPase-activating protein for Ypt6, specifically interacts with Ypt32 as an effector. Taken together with the previously proposed Rab cascade within the Golgi, we propose that multiple Rab cascades interact at the intersection of secretory and endosomal pathways and play significant roles in traffic within and around the Golgi apparatus.
Abstract
The Golgi apparatus functions as the central station of membrane traffic in cells, where newly synthesized proteins moving along the secretory pathway merge with proteins recycled from subsequent membrane organelles such as endosomes. A series of Rab GTPases act consecutively and in concert with the maturation of cis- to-trans cisternae of the Golgi apparatus. Rab GTPases control various steps in intracellular membrane traffic by recruiting downstream effector proteins. Here, we report the dynamics of Ypt6, a yeast member of the Rab GTPase family, which mediates the fusion of vesicles from endosomes at the Golgi apparatus. Ypt6 resides temporarily at the Golgi and dissociates into the cytosol upon arrival of Ypt32, another Rab GTPase functioning in the late Golgi. We found that Gyp6, a putative GTPase-activating protein (GAP) for Ypt6, specifically interacts with Ypt32, most likely as an effector. Disruption of GYP6 or introduction of a Rab–GAP activity-deficient mutation in GYP6 resulted in continual residence of Ypt6 at the Golgi. We propose that Ypt32 acts to terminate endosome-to-Golgi traffic through a Rab–GAP cascade as it does for cis-to-trans intra-Golgi traffic. Simultaneous disruption of GAP for early-acting Rab proteins in the Golgi showed appreciable defects in post-Golgi trafficking, but did not significantly affect cell growth.
Rab GTPase cycles between the active, membrane-bound form and the inactive, cytosolic form via the action of intrinsic GTP exchange factor (GEF) and GTPase-activating protein (GAP). Once Rab GTPase is activated and targeted to the membrane, various downstream effectors are recruited onto the membrane to fulfill various intracellular membrane trafficking processes (1). Each Rab GTPase exhibits a unique spatiotemporal localization pattern at a particular membranous compartment to precisely regulate vesicular traffic. Thus, Rab GTPase and its effectors define a compartment’s identity (2, 3).
The Rab–GEF cascade consists of sequential regulation of Rab GTPases in which an upstream Rab recruits a GEF for downstream Rab activation. This continuous activation of Rab proteins occurs at several organelles such as endosomes, transport vesicles, and the Golgi apparatus. For example, Rab5 recruits the Mon1–Ccz1 complex, which is a GEF for Rab7 and facilitates Rab5-Rab7 conversion during early to late endosome maturation in yeast and animal cells (4, 5). In the exocytic pathway of the budding yeast Saccharomyces cerevisiae, transition from Ypt1 to Ypt31/32 occurs at the Golgi apparatus by recruitment of Ypt31/32 GEF as a Ypt1 effector (6). Once Ypt31/32 is activated, Sec2, a GEF for Sec4, is recruited onto the membrane to activate Sec4 at post-Golgi transport vesicles (7). Although the Rab–GEF cascade regulates the continuity of different Rab GTPases in such organelles (1), it also potentially creates instability because continuous activation of early Rab GTPase leads to the concomitant existence of two Rab proteins on the same membrane. Recent work has provided evidence for a Rab–GAP cascade, in which a GAP of a former Rab is the effector of subsequent Rab proteins at the Golgi apparatus in yeast (8). Termination of early acting Ypt1 is facilitated by the recruitment of Gyp1, a GAP for Ypt1, through binding to Ypt32 as an effector at a single cisterna of the Golgi.
This counter current activity of Rab GTPases could be a main mechanism of compartment maturation (9, 10). Because Rab defines the identity of a membrane compartment through the recruitment of effector proteins, the transition from one Rab to another changes the properties of the membrane where they exist. In the Golgi apparatus, the disruption of the boundary between two Rab proteins would affect cisternal maturation. This appears to be indeed the case in yeast in the transition of an early acting Rab-containing compartment to another containing a late-acting Rab (8). Nevertheless, Gyp1, a GAP responsible for the Rab–GAP cascade in the Golgi, is dispensable for yeast cell growth. Other mechanisms may exist, in which transient contact between two compartments contributes to the maturation of the Golgi apparatus (11).
Ypt6, the yeast counterpart of Rab6, has been reported to associate with the Golgi apparatus and regulate the fusion of vesicles from endosomes (12). In mammalian cells, accumulating evidence suggests that Rab6 localizes at the trans-Golgi region and functions in retrograde protein transport and in Golgi structural organization (13, 14). Ypt6 may very well function in the yeast Golgi maintenance. Ypt6 is activated in to the GTP-bound state by the Ric1–Rgp1 GEF complex and associates with the Golgi membrane, where it recruits a tethering factor called the Golgi-associated retrograde protein (GARP) complex (15, 16). S. cerevisiae has eight Rab GAP proteins containing the conserved Tre2-Bub2-Cdc16 (TBC) domain, which provides the catalytic activity toward Rab GTPases. Putative GAP proteins for Ypt6, including Gyp6, have been identified and characterized in vitro (17, 18). However, TBC domain-containing proteins have broad GAP activities toward multiple Rab proteins in contrast to the high specificity between Rab–GEF pairs (19). In the case of Ypt6, not only Gyp6 but also Gyp2 and Gyp8 show in vitro GAP activity toward Ypt6 (20, 21). Thus, the precise mechanisms controlling Ypt6 dynamics and the relationship between multiple Rab GTPases in the Golgi apparatus remain obscure.
Here, we report roles of Rab–GAP in regulation of Ypt6 dynamics in S. cerevisiae. We have found that Ypt6 resides at the medial-Golgi and dissociates from the membrane at the onset of Ypt31/32 arrival during maturation. Gyp6, a putative GAP for Ypt6, binds to Ypt32 and is recruited to the membrane, facilitating the dissociation of Ypt6. This counteracting regulation of Ypt6 requires the GAP activity of Gyp6, suggesting that a Rab–GAP cascade defines the membrane traffic from the endosomes to the Golgi apparatus.
Results
Ypt6 Resides in the Boundary Between the Cis- and Trans-Golgi.
Rab–GEF and –GAP cascades have been proposed to regulate the sequential activation and deactivation of the two Rab GTPases, Ypt1 and Ypt31/32, within the Golgi apparatus (6, 8). However, the regulation of another Rab GTPase, Ypt6, which is a Rab6 homolog and mediates the fusion of vesicles from endosomes to the Golgi apparatus, is obscure. Because Golgi cisternae in S. cerevisiae are unstacked and dispersed in the cytoplasm (22), the timing and duration of Ypt6 recruitment to the Golgi can be efficiently estimated by measuring the colocalization with known Golgi cisternae markers. We analyzed the extent of colocalization of Rab GTPases with cis- and trans-Golgi resident proteins, cis-Golgi localized SNARE protein Sed5 and the GEF of Arf GTPase Sec7, respectively (23). The percentages of mRuby-Sed5 colocalized with each Rab GTPase were 80% for GFP-Ypt1, 54% for GFP-Yp6, and 10% for GFP-Ypt32 (Fig. 1 A and B and Table S1), whereas those of Sec7-mRuby signals colocalized with Rab GTPases were 49% for GFP-Ypt1, 58% for GFP-Yp6, and 87% for GFP-Ypt32 (Fig. 1 C and D and Table S1). This result reflects the temporal occupation of Rab GTPases within the Golgi apparatus, as Ypt1 resides at the cis-Golgi whereas Ypt31/32 functions in later compartments including trans-Golgi cisternae and the trans-Golgi network (TGN). Thus, the intermediate value of colocalization for Ypt6 suggests that it resides either in the boundary between the cis- and trans-Golgi or in both compartments. The high degree of colocalization between GFP-Ypt1 and Sec7-mRuby (49%) confirms that Ypt1 can stay in later compartments of the Golgi apparatus as reported (24).
Distinct localization of Rab GTPases at the Golgi. (A) Localization of Rab GTPases with the cis-Golgi marker mRuby-Sed5. Wild-type cells expressing GFP-tagged Rab GTPases are indicated and mRuby-Sed5 were grown to a midlogarithmic phase in synthetic medium at 30 °C and observed by 3D confocal fluorescence microscopy. Dashed lines indicate the edge of the cell. Filled arrowheads indicate colocalization whereas open arrowheads point to GFP-Rab GTPase puncta that do not show mRuby-Sed5 signal. (Scale bar, 2 µm.) (B) Bar graph showing the percentage of mRuby-Sed5 spots colocalized with each of the three GFP-tagged Rab GTPases. Error bars indicate SE from at least 15 independent cells. (C) Localization of Rab GTPases with trans-Golgi marker Sec7-mRuby. Wild-type cells expressing GFP-tagged proteins are indicated and Sec7-mRuby were grown and observed as in A. (D) Bar graph shows the percentage of Sec7-mRuby signals colocalized with each of the three GFP-tagged proteins. Error bars indicate SE from at least 15 independent cells.
Mammalian Rab6 has been shown to localize and function in the TGN for the fusion of endosome-derived vesicles (13). However, Ypt6 does not appear to localize exclusively to TGN, because more than 50% of GFP-Ypt6 fluorescence overlaps with the cis-Golgi marker Sed5. We decided to compare the relationship between these Rab GTPases within the Golgi apparatus by direct colocalization analysis of GFP-Ypt6 with mRFP-Ypt1 and mRFP-Ypt32. As shown in Fig. 2 (WT) and Table S1, GFP-Ypt6 colocalized well with mRFP-Ypt1 (63.0 ± 4.64%), but almost not at all with mRFP-Ypt32 (8.2 ± 1.90%). This result suggests that Ypt1 and Ypt6 function in the early Golgi, and that the roles of Ypt6 and Ypt32 are only partially overlapping or may even be exclusive in the late Golgi.
Deletion of GYP6 leads to colocalization of GFP-Ypt6 with mRFP-Ypt32. Wild-type (WT), gyp1, and gyp6 cells expressing indicated pairs of either GFP- or mRFP-tagged Rab GTPases were grown to a midlogarithmic phase and observed by 3D confocal fluorescence microcopy. Dashed lines indicate the edge of the cell. Open and filled arrowheads indicate mRFP-Ypt32 with or without GFP-Ypt6 signals, respectively. (Scale bar, 2 µm.)
The Rab–GAP cascade has been proposed to execute spatiotemporal regulation of Rab GTPases in the Golgi apparatus (8). The unique intra-Golgi localization of Ypt6 tempted us to investigate the role of Rab–GAPs in the Ypt6 localization. In the case of Ypt1 and Ypt31/32, the boundary has shown to be maintained by a Rab–GAP cascade, and this boundary is lost when Gyp1, the Rab–GAP for Ypt1, is disrupted (8). As shown in Fig. 2 (gyp1) and Table S1, deletion of Gyp1 did not affect the relationships between GFP-Ypt6 and mRFP-Ypt1 (71.0 ± 2.93%) and between GFP-Ypt6 and mRFP-Ypt32 (11.6 ± 3.56%). However, disruption of Gyp6, a putative GAP for Ypt6, caused a drastic change. We observed significant colocalization of GFP-Ypt6 puncta with mRFP-Ypt32 (51.0 ± 0.04%), especially at incipient bud sites, bud tips, and mother-bud neck regions where mRFP-Ypt32 concentrates (Fig. 2, gyp6). The relationship between GFP-Ypt6 and mRFP-Ypt1 (53.5 ± 3.61%) was not markedly affected in gyp6 cells. These results indicate that Gyp6 plays a critical role in mutually exclusive localization of Ypt6 and Ypt32.
Next, we investigated whether GYP6 disruption might extend the existence of Ypt6 or cause early arrival of Ypt32 to the Golgi apparatus. For this purpose, we took a 3D time-lapse fluorescence measurement method (4D imaging) by superresolution confocal live imaging microscopy (SCLIM) (25–28) and analyzed the dynamics of the Rab GTPases in detail. After deconvolution and projection into 2D, we set several appropriate areas of interest (shown as open squares in Fig. 3) and compared total fluorescence intensities of either GFP-Ypt6 or GFP-Ypt32 with that of the trans-Golgi/TGN resident protein Sec7-mRFP. In wild-type cells, GFP-Ypt6 signals began to decrease before the Sec7-mRFP signal reached the peak (Fig. 3A and Fig. S1 A and C). In gyp6 cells, in contrast, concomitant decrease of both GFP-Ypt6 and Sec7-mRFP signals was observed over time (Fig. 3A and Fig. S1 B and C). GFP-Ypt32 signals showed up in the Sec7-positive structure and reached peak just before the disappearance of the Sec7-mRFP signal (Fig. 3B and Fig. S2 A and B). No significant difference was observed between wild-type and gyp6 cells (Fig. S2C). These data suggest that Gyp6 has a role in dissociation of Ypt6 from the Golgi earlier than the arrival of Sec7, but does not in later recruitment of Ypt32 to the membrane.
Four-dimensional live imaging reveals the dissociation of Ypt6 from the Golgi at the onset of Ypt32 arrival. (A) Behaviors of GFP-Ypt6 and Sec7-mRFP signals in chosen areas of interest (open squares) from the movies (Upper, Movie S1, wt; Lower, Movie S2, gyp6). Dashed lines indicate the edge of the cell. Lower montages show time-lapse images of the indicated squares. Graphs indicate relative fluorescence intensities of each fluorescent signal of the region shown in the montage over time. (B) Dynamics of GFP-Ypt32 and Sec7-mRFP from the movies (Upper, Movie S3, wt; Lower, Movie S4, gyp6) as in A. (Scale bar, 2 µm.) RFI, relative fluorescence intensity.
Gyp6 Localizes at the Trans-Golgi/TGN and Interacts with Ypt32.
To determine the localization of Gyp6 in the Golgi, we expressed Gyp6-GFP and compared with Sec7-mRFP and mRFP-Ypt32. Gyp6-GFP puncta showed good colocalization with both Sec7-mRFP (96.4 ± 1.49%) and mRFP-Ypt32 (64.1 ± 6.29%), indicating its trans-Golgi/TGN localization (Fig. 4A and Table S1). In a ypt32 temperature-sensitive mutant (ypt32ts in the ypt31Δ background), Gyp6-GFP no longer showed clear residence on trans-Golgi/TGN marked by Sec7-mRFP, even at the permissive temperature (Fig. 4B). Because the total amount of Gyp6-GFP protein was not reduced (Fig. 4C), this observation indicates that the Gyp6-GFP localization to the Golgi apparatus is Ypt32-dependent.
Gyp6 resides in the Golgi and interacts with Ypt32 as a putative effector. (A) Colocalization of Gyp6-GFP with Sec7-mRFP. Wild-type cells expressing Gyp6-GFP or Gyp6R155K-GFP and either Sec7-mRFP or mRFP-Ypt32 were grown to a midlogarithmic phase, and imaged by 3D confocal fluorescence microscopy. Dashed lines indicate the edge of the cell. (Scale bar, 2 µm.) (B) ypt32 temperature-sensitive mutant cells (ypt32ts) expressing Gyp6-GFP and Sec7-mRFP were grown at 25 °C to a midlogarithmic phase and imaged by fluorescence microscopy as in A. (C) Total lysates of wild-type and ypt32ts cells expressing Gyp6-GFP after incubation either at 25 °C or 37 °C for 30 min were separated by SDS/PAGE followed by immunoblotting analysis using anti-GFP antibodies. Dolichol phosphate mannose synthase, Dpm1, was also probed as a control. (D) Yeast two-hybrid assay between GYP6 and Rab GTPase alleles of wild-type GTP- and GDP-fixed states. Growth on the plate lacking tryptophan/leucine (WL) indicates the cells harboring plasmids. Growth on the plate lacking tryptophan/leucine/histidine (WLH) with or without 5 mM 3-AT indicates a positive interaction. (E) Pull-down of Gyp6-3HA and Gyp1-3HA by GST-tagged Ypt32. Lysates from yeast cells overexpressing Gyp6-3HA or Gyp1-3HA were incubated with glutathione Sepharose preincubated with either GST- or GST-Ypt32 in the presence of GTPγS. Samples were separated by SDS/PAGE and signals were detected by immunoblotting using an antibody against HA. One-percent input of yeast total lysate was run as a control.
To test the interaction of Gyp6 with Ypt32, we used a yeast two-hybrid system. GYP6 showed positive interaction with YPT32 in either the wild-type or the GTP-fixed (Q79L) form (Fig. 4D). Weak interaction of GYP6 with YPT6 was observed, confirming its very low affinity for the substrate Ypt6 (29). We also found similar positive interaction of SEC2 with YPT32, confirming the specificity of each interaction (Fig. 4D). We could not observe positive interaction of GYP6 with YPT31, the functionally redundant gene homolog of YPT32 (30), raising the possibility that these two Rab11 homologs may be functionally diversified. Nevertheless, no perturbation of GFP-Ypt6 dynamics was observed in the ypt32 single mutant (Fig. S3), indicating that Ypt31 compensates for the Ypt32 defect in the recruitment of Gyp6 to a certain extent. We next performed pull-down experiments with the yeast lysates expressing Gyp6-3HA using purified recombinant GST-Ypt32 to confirm their interaction (Fig. 4E). Gyp6-3HA was brought down with GST-Ypt32 but not with GST alone. A similar result was obtained for Gyp1-3HA, which has been shown to interact with Ypt32 (8). The low level of affinity might imply that the interaction between Gyp6 and Ypt32 is transient. In summary, these experiments demonstrate that Ypt32 recruits Gyp6 to the Golgi apparatus when it is in the active GTP-state. In other words, Gyp6 can be considered as a putative effector of Ypt32.
Gyp6 belongs to the TBC domain-containing family of GAPs for Rab GTPases (17). We made the deletion construct of GYP6 to see whether the observed Gyp6 interaction with Ypt32 is mediated by the catalytic TBC domain. C-terminal deletion of Gyp6, which contains the intact TBC domain, abolished both Gyp6 localization at the Golgi and mutually exclusive localization of Ypt6 and Ypt32 (Fig. S4 A and B and Table S1). No significant alteration for the C-terminal deletion mutant was observed on the level and the stability of the protein (Fig. S4C). Yeast two-hybrid analysis confirmed that the C-terminal region, which is distinct from the catalytic TBC domain, is responsible for the interaction with Ypt32 (Fig. S4D).
GAP Activity of Gyp6 Is Important to Regulate Ypt6 Dynamics in the Golgi Apparatus.
An arginine residue within the TBC domain is essential for GAP activity toward Rab GTPases and is a conserved feature of the TBC family proteins (31). In the case of Gyp6, replacement of the arginine residue in the TBC domain was shown to cause the loss of GAP activity toward Ypt6 (29). To determine whether the GAP activity of Gyp6 is important to control Ypt6 dynamics in the Golgi membrane, we constructed the substitution Arg-155 to Lys (R155K) of Gyp6 tagged with GFP and examined its subcellular localization. As shown in Fig. 4A and Table S1, the observed pattern of Gyp6R155K-GFP was indistinguishable from that of Gyp6-GFP. No detectable defect was observed for the R155K mutant on the total level and stability of Gyp6 protein (Fig. 5C). To examine the effect of the R155K mutation of Gyp6 on the dissociation of Ypt6 from the Golgi membrane, we examined GFP-Ypt6 dynamics relative to Sec7-mRFP by 4D imaging using SCLIM, in the gyp6 cells expressing either Gyp6-3HA or Gyp6R155K-3HA. As shown in Fig. 5A and Fig. S5 A and C, GFP-Ypt6 dissociation occurred in a similar fashion to the wild type in Gyp6-3HA cells, confirming that the Gyp6-3HA construct was functional. However, in the Gyp6R155K-3HA–expressing cells, GFP-Ypt6 failed to reduce the signals before the signal reduction of Sec7-mRFP (Fig. 5B and Fig. S5 B and C). These observations demonstrate that the GAP activity of Gyp6 is critical for the dissociation of Ypt6 during the course of Golgi maturation.
Four-dimensional live imaging demonstrates the requirement of GAP-activity of Gyp6 for the dissociation of Ypt6 from the Golgi. (A and B) Behaviors of GFP-Ypt6 and Sec7-mRFP from the movies (Upper, Movie S5, GYP6-3HA; Lower, Movie S6, gyp6R155K-3HA). Dashed lines indicate the edge of the cell. Lower montages show time-lapse images of the areas marked by squares. Graphs indicate relative fluorescence intensities of each fluorescent signal of the region shown in the montage over time. (Scale bar, 2 µm.) (C) The steady-state levels of wild-type and R155K mutant Gyp6-3HA. The amount of Gyp6-3HA was examined by Western blot analysis of the total cell lysates using anti-HA monoclonal antibody. Dpm1 was used as a control.
Failure of Rab Transition Perturbs Post-Golgi Membrane Trafficking, but Does Not Significantly Affect the Cisternal Maturation of the Golgi.
The Rab conversion has been proposed as a key characteristic of compartment maturation (4, 32, 33). We examined whether the cisternal maturation of the Golgi is affected by the disruption of the boundary between Golgi-resident Rab GTPases, by looking at the gyp1 gyp6 double mutant. In gyp1 gyp6 cells, Ypt1, Ypt6, and Ypt32 appear to more or less colocalize in the Golgi (Fig. 6A and Table S1). This is a clear piece of evidence confirming the disruption of the boundary between the Rab GTPases. To estimate the rates of maturation, we marked Golgi cisternae with GFP-Gos1 (medial-Golgi) and Sec7-mRFP (trans-Golgi) (23), and then measured the ratio of the red/green fluorescence transition and calculated its doubling time. The color transition of cisternae in wild-type cells had a ratio doubling time of 22.5 ± 8.3 s (±SD; n = 13), whereas in gyp1 gyp6 cells it was ∼34% increased compared with wild-type cells, 30.1 ± 5.9 s (n = 13) (Fig. 6B). Statistically, the difference of these values was not significant (Student t test, P > 0.05). These observations suggest that efficient transition of these Rab GTPases at the Golgi is not critical but may be important for the speed of maturation.
Simultaneous deletion of GYP1 and GYP6 does not affect the Golgi cisternal maturation. (A) gyp1 gyp6 cells expressing indicated pairs of either GFP- or mRFP-tagged Rab GTPases were grown to a midlogarithmic phase and observed by 3D confocal fluorescent microcopy. Dashed lines indicate the edge of the cell. (Scale bar, 2 µm.) (B) A box plot representing the distribution of the ratio-doubling time for the color conversion of cisternae from GFP-Gos1 to Sec7-mRFP in wild-type and gyp1 gyp6 cells. The horizontal line in each box represents the median value of the distribution. The boundaries of a box represent the lower and upper quartile values. The whiskers extending vertically from the upper and lower portions of each box represent the extent of the rest of the data. Student t test P value was 0.12 in comparison between the wild type and the gyp1 gyp6 double mutant.
We also analyzed protein recycling and export in gyp1 gyp6 cells. Snc1 cycles between post-Golgi organelles and the plasma membrane and primarily localizes to the plasma membrane (34). The localization of GFP-Snc1 was normal in gyp6 cells, whereas accumulation of internal structures was seen in gyp1 and gyp1 gyp6 cells (Fig. S6A), suggesting recycling defects in these mutants. We also observed mislocalization of Vps10, a sorting receptor for vacuolar hydrolases, which cycles between the late-Golgi and prevacuolar endosome-like compartments (PVCs) (35). Vps10-3xGFP was found to be in punctate structures in wild-type and gyp6 cells as reported previously. In gyp1 and gyp1 gyp6 cells, Vps10-3xGFP was mislocalized to the vacuolar membrane and occasionally to perivacuolar dot-like structures (Fig. S6B). The dot-like structures are similar to the exaggerated PVC-like structures characteristic of the mutants that are defective in the protein export from the PVC, also suggesting recycling defects. Colony blot analysis (36) indicated gyp1, gyp6, and gyp1 gyp6 cells showed weak but detectable missecretion of caboxypeptidase Y (Fig. S6C). Additionally, activity-staining gel electrophoresis indicated that invertase is underglycosylated in gyp1 and gyp1 gyp6 mutants (Fig. S6D). These results indicate that the perturbation of the Rab regulation at the Golgi appreciably affected the Golgi functions but was not deleterious to the cells.
Discussion
Intracellular logistics based on membrane traffic is tightly regulated to ensure cellular homeostasis. Rab GTPases play pivotal roles in regulation of multiple traffic events (1). They show strict localization when they are in the active GTP state and thus are believed to define the identity of compartments (2, 3). The differentiated roles of Rab GTPases in the Golgi apparatus are of particular interest because the mechanism of compartmentalization of the Golgi is still a question of dispute. High-resolution live imaging is a very powerful approach to address this question, and the budding yeast S. cerevisiae has been used as an advantageous organism for this purpose because its Golgi cisternae are unstacked and easy to resolve by fluorescence microscopy (22, 37). Cisternae of the Golgi change their identity from cis to trans nature by maturation (23, 38). Regarding Rab GTPases, Ypt1 functions in cis-Golgi cisternae and Ypt31/32 reside in trans-Golgi cisternae/TGN, and the efficient transition from early-acting Ypt1 to subsequent Ypt31/32 is fulfilled by the action of Rab–GEF and –GAP cascades (8). In the present paper, we demonstrate that another Rab–GAP cascade is present in the yeast Golgi apparatus in the transition from Ypt6 at the medial-Golgi to Ypt31/32 at the trans-Golgi/TGN.
Ypt6 Resides at the Medial-Golgi During Maturation.
Ypt1 functions in the fusion of ER-derived vesicles with the Golgi at cis-Golgi cisternae (39, 40), whereas Ypt31/32 locate at the trans-Golgi/TGN and regulate various traffic events directly and indirectly, e.g., fusion of vesicles from endosomes (41), movement of the Golgi apparatus (42), and subsequent Sec4 activation through recruitment of Sec2, a GEF for Sec4 (7). Ypt6 is the yeast homolog of the mammalian Rab6, which functions in the fusion of endosome-derived vesicles to the Golgi by recruiting the GARP complex as its effector (12, 15, 16). In mammals and plants, it is generally agreed that Rab6 functions in trans-Golgi/TGN (13, 14). However, our results showed significant colocalization of GFP-Ypt6 with mRuby-Sed5, suggesting that Ypt6 also localizes to cis-Golgi cisternae in yeast. Colocalization of GFP-Ypt6 with Sec7-mRFP ensures that Ypt6 does localize to trans-Golgi/TGN, however, GFP-Ypt6 was scarcely colocalized with the trans-Golgi/TGN-localized Rab GTPase Ypt32. Live imaging of the dynamics of GFP-Ypt6 and GFP-Ypt32 indicated that Ypt6 dissociates from the Golgi at the onset of Ypt32 arrival. Together, our data suggest that Ypt6 resides and most probably functions in the medial-Golgi. Ypt6 and Ypt31/32 are both reported to function in the fusion of vesicles that contain diverse sets of cargo proteins such as Snc1 and Vps10 from endosomes (12, 34, 41, 43). Budding yeast might have used a temporal hierarchy between these Rab GTPases for strict sorting of a diverse set of cargo proteins upon their arrival at the Golgi.
Ypt32 Recruits Gyp6 to the Golgi as a Putative Effector.
We have shown that Gyp6-GFP colocalizes with trans-Golgi resident proteins, Sec7-mRFP and mRFP-Ypt32. This is inconsistent with a previous study claiming the presence of Gyp6 in the endosomal/prevacuolar compartment (44). Considering the role of Gyp6 as a Ypt6 GAP, its Golgi localization looks reasonable. The Golgi localization of Gyp6 is lost when the Ypt31/32 function is compromised by a temperature-sensitive mutation. This indicates that Ypt31/32 is important for the localization of Gyp6. The interaction between Ypt32 and Gyp6 has been confirmed by yeast two-hybrid assay and pull-down experiments, although the nucleotide-dependent interaction between Ypt32 and Gyp6 has not been detected in vitro. The C-terminal region of Gyp6, which is distinct from the catalytic TBC domain, is responsible for the interaction. The GTP form of Ypt32 appears to be preferred in this interaction in vivo, while no detectable GAP activity has been reported for Gyp6 toward Ypt32 in vitro (19). We have also detected weak interaction between Gyp6 and Ypt6 by yeast two-hybrid, confirming a low affinity of Gyp6 toward Ypt6 in vitro (29). These observations strongly suggest that Ypt32 binds Gyp6 as a putative effector.
The Rab–GAP Cascade Between Ypt6 and Ypt31/32.
We show that the deletion of GYP6 leads to the coexistence of Ypt6 and Ypt32 in the Golgi, particularly at the bud neck. This unusual situation is probably caused by the continual activation of Ypt6 in the post-Golgi transport vesicles that accumulate at the site of cytokinesis. Among eight TBC-domain containing putative GAP proteins in yeast (17, 18), only the GYP6 deletion resulted in such a defect in our analysis. Gyp2 was also reported as a possible GAP for Ypt6 (20, 45), but the gyp2 mutant did not show any defect in Ypt6 localization (Fig. S7). Deletion of GYP1 did not perturb the mutually exclusive localization between Ypt6 and Ypt32 either. This is in agreement with the endocytic events in which deletion of Msb3/Gyp3 effects only the localization of Vps21 (19). Taken together, the Rab–GAP substrate specificity might be much more rigorous than what has been predicted from in vitro binding experiments.
Our live-cell imaging analysis by SCLIM has demonstrated that Gyp6 is essential for temporal occupation of Ypt6 at the Golgi, probably at the medial cisternae. The conserved arginine residue in the TBC domain of Gyp6, which is reported important for its GAP activity toward Ypt6 (29), is critical for this role of Gyp6. Taking all these data together, we conclude that a Rab–GAP cascade exists between Ypt6 and Ypt32.
Localization of Ypt1 and Ypt32 overlaps in the Golgi (8), but that of Ypt6 and Ypt32 is exclusive. Presence of a Rab–GEF cascade for Ypt1/Ypt32 may explain this difference. In mammalian cells, a putative Rab–GEF cascade has been proposed between medial-Golgi-localized Rab33B and the trans-Golgi protein Rab6A via recruitment of Ric1, a component of the GEF complex for Rab6 (14, 46). Whether a similar mechanism might also exist in yeast or not awaits further investigation.
Continual Activation of Multiple Rab Proteins at the Golgi Has only a Slight Effect on Golgi Maturation.
We found that the perturbation of Rab–GAP cascade affects normal Golgi function. The simultaneous deletion of Gyp1 and Gyp6, which strongly affected the normal localization of the three Golgi Rab GTPases in yeast, resulted in some appreciable transport defects of both secretory and recycling cargoes. Our results suggest that coordinated expression of the Rab GTPases is indeed important for the Golgi function.
Recent studies suggest that mammalian Rab6 is important not only for membrane traffic but also for organization and maintenance of the Golgi (13, 14). To examine whether concurrent existence of the Golgi Rab GTPases affects the maintenance of the Golgi structure and/or maturation of cisternae, we constructed a gyp1 gyp6 double mutant, in which early-acting Ypt1 and Ypt6 are not eliminated and thus coexist with the later-acting Ypt31/32 in the Golgi. Detailed analysis by 4D fluorescent microscopy using SCLIM shows that the maturation speed of the Golgi in gyp1 gyp6 mutant cells is slightly affected, but not significantly different from that of the wild type. Our observation might imply the presence of additional fail-safe mechanism(s) that ensures the Golgi compartmentalization and maturation.
Materials and Methods
Experimental details are mostly described in SI Materials and Methods.
Yeast Strains and Media.
Standard methods and media were used (47). The strains used in this study are listed in Table S2. All strains in this study, except AH109 for yeast two-hybrid assay, were from a YPH499 strain background (48). Gene disruption and insertion were basically done by PCR-mediated gene replacement (49, 50) and verified by PCR or phenotype.
Acknowledgments
We thank Kalai Madhi Muniandy for technical support and all members of the A.N. laboratory for helpful comments. This work was supported by a Grant-in-Aid for Specially Promoted Research (Grant 20001009 to A.N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Extreme Photonics and Cellular Systems Biology Projects of RIKEN (The Institute of Physical and Chemical Research) (Japan) (A.N.).
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. S.R.P. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1308627110/-/DCSupplemental.