Abstract

Ypt–Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt–Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport^{1, 2, 3}. An interesting possibility is that Ypt–Rabs coordinate separate steps of the transport pathways⁴. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5–7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi^{8, 9, 10}. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors^{11, 12, 13}. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120–Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.

Introduction

We have previously shown that the multisubunit TRAPP complex acts as a GEF for Ypt1 and Ypt31 GTPases⁵. Here, we used two independent methods to show that the TRAPPII-specific subunits Trs120 and Trs130 are required for the Ypt31 GEF activity, whereas they inhibit the Ypt1 GEF activity of TRAPP. First, TRAPP purified from trs120- or trs130-mutant cells had enhanced GEF activity on Ypt1, but did not act as a Ypt31/32 GEF. Second, wild-type TRAPPI and TRAPPII complexes were separated on a sizing column and their specificity as GEFs for Ypt1 and Ypt31, respectively, was determined.

Two types of trs130 mutation were used for the first approach, trs130ts and trs130. The trs130ts allele encodes a protein that lacks the 33 carboxy-terminal amino acids, and confers a temperature-sensitive growth phenotype¹⁰. The chromosomal gene encoding the Trs130 protein was tagged at the C-terminus with HA in wild-type and trs130ts-mutant cells¹³. Immunoblot analysis of wild-type and trs130ts-mutant cell lysates revealed that the level of mutant Trs130ts–HA protein is barely detectible (Fig. 1a), suggesting that it is unstable. The trs130 allele does not express any Trs130 protein and is suppressed by overexpression of Ypt31 to allow cell viability. In this mutant strain, Trs120, the other TRAPPII essential subunit, was tagged at the C-terminus on the chromosome with Myc. Immunoblot analysis of Trs120–Myc in wild-type and trs130 mutant cell lysates revealed that the level of Trs120–Myc protein is normal in mutant cells (Fig. 1b). This enabled determination of the role of Trs130 independently of Trs120.

Figure 1: Purification of GST–Bet5-associated complexes from wild-type (WT) and trs130- and trs120-mutant cells.

(a) GST–Bet5-associated complex purified from trs130ts-mutant cells contains low level of Trs130 protein. Left: the level of Trs130 protein is markedly reduced in trs130ts-mutant cell lysates. Lysates from wild type (VSY459) cells and from trs130ts (VSY446) cells, in which the chromosomal copy of the TRS130 gene was tagged with HA, were tested by immunoblot analysis. Right: The GST–Bet5-associated complex purified from trs130ts-mutant cells contains a low level of Trs130 protein. GST or GST–Bet5 was pulled-down from wild-type and trs130ts-mutant cell lysates using GA-beads. Equal amounts (as determined by Bradford assay) of cell lysates (5 g; left), or purified GST complexes (0.2 g; right), were subjected to immunoblot analyses using the following antibodies: anti-HA for Trs130–HA; anti-GST for GST and GST–Bet5; and anti-G-6-PDH for equal loading control. (b) GST–Bet5-associated complex purified from trs130-mutant cells contains normal levels of Trs120–Myc protein. Lysates (left) and GST–Bet5-associated complex (right) from wild-type + 2 Ypt31 (NSY1157) and trs130 + 2 Ypt31 (VSY454) cells, in which the chromosomal copy of TRS120 was tagged with Myc, respectively, were prepared and tested as in a, except that anti-Myc antibodies were used for detecting the Trs120–Myc, and anti-Ypt31/32 antibodies were used for detecting Ypt31. (c) GST–Bet5-associated complex purified from trs120-mutant cells contains a low level of Trs130 protein. Left: The level of Trs130 protein is markedly reduced in trs120-mutant cell lysates. Lysates from wild-type + 2 Ypt31 (NSY1158), and trs120 + 2 Ypt31 (VSY451) cells, in which the chromosomal copy of the TRS130 gene was tagged with Myc, were tested by immunoblot analysis. Right: GST–Bet5-associated complex purified from trs120-mutant cells contains a low level of Trs130 protein. GST or GST–Bet5 was pulled-down from wild-type and trs120-mutant cell lysates using GA-beads. Equal amounts (as determined by Bradford assay) of cell lysates (5 g; left), or purified GST complexes (0.2 g; right), were subjected to immunoblot analyses using the same antibodies as in b. Results shown in this figure are representative of at least two independent experiments.

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Bet3–Bet5-associated complexes were characterized as TRAPP in two independent studies using different tags^{8, 14}. Bet3 and Bet5 are two essential TRAPPI/II subunits, and we have previously used GST-tagged Bet3 and Bet5 to purify TRAPP complexes. Overexpression of Bet3 is detrimental in trs130-mutant cells (see below). Therefore, we used GST–Bet5 to purify TRAPP complexes in the experiments described below; these complexes should include both TRAPPI and TRAPPII. Accordingly, the protein profile of the GST–Bet5-associated complex shows proteins with molecular weights corresponding to all ten TRAPPI and TRAPPII subunits (see Supplementary Information, Fig. S1b).

GST–Bet5, or GST as a negative control, were expressed in yeast cells, and were purified by precipitating with glutathione–agarose beads, followed by elution with glutathione. GST and GST–Bet5-associated complexes were tested for the presence of Trs130–HA or Trs120–Myc protein using immunoblot analysis. Wild-type Trs130–HA protein coprecipitated with GST–Bet5 (but not with GST). As expected from the low level of Trs130ts–HA protein in trs130ts mutant cell lysates, very little of this protein is present in the GST–Bet5-associated complexes (Fig. 1a). GST–Bet5-associated complexes (but not GST) purified from wild-type and trs130-mutant cells contain similar levels of Trs120–Myc protein (Fig. 1b). This result implies that Trs130 is not required for the attachment of Trs120 to TRAPP. These GST–Bet5-associated complexes were used for GEF assays.

TRAPP purified from wild-type cells using GST–Bet5/Bet3 was previously shown to have Ypt1 and Ypt31 GEF activities using GDP-loss and GTP-uptake assays⁵. The GST–Bet3-associated complex did not have a Sec4 GEF activity. To verify the Ypt specificity of the GST–Bet5-associated complex, we tested its activity on Sec4. GST–Sec2 served as a positive control for this experiment, and it stimulated GDP release from Sec4. In contrast, GDP release from Sec4 was not stimulated by GST–Bet5-associated complex above the background of GST alone (see Supplementary Information, Fig. S1c).

Equal amounts of purified GST–Bet5-associated complexes, from wild-type, trs130ts- or trs130-mutant cells, were used in GDP-loss assays with Ypt1 or Ypt31 as a substrate. We expected that TRAPP purified from trs130-mutant cells would have Ypt1 GEF activity, because Trs130 is present in the trans-Golgi, but not in the cis-Golgi¹⁵, where Ypt1 functions⁶. Importantly, the trs130-mutant TRAPP complex had a significantly higher Ypt1 GEF activity than that of the wild type (Fig. 2a, b). These results indicate that trs130-mutant TRAPP acts as a Ypt1 GEF, functioning even better than wild-type TRAPP. If Trs130 protein is required for the Ypt31 GEF activity of TRAPP, as suggested by the genetic interactions^{11, 12, 13}, then the trs130ts-mutant TRAPP complex, which almost lacks Trs130 (Fig. 1a), or trs130 TRAPP, should be defective for this activity. Indeed, whereas GST–Bet5-associated complex purified from wild-type cells contained robust Ypt31 GEF activity, no activity was detected in TRAPP purified from trs130-mutant cells above the GST pulldown background (Fig. 2a, b). These results suggest that Trs130 is required for the Ypt31 GEF activity of TRAPP.

Figure 2: TRAPP purified from trs130- and trs120-mutant cells is defective in Ypt31 GEF, but possesses higher Ypt1 GEF activity than wild-type TRAPP.

(a) GST–Bet5-associated complex from trs130ts-mutant cells has increased Ypt1 GEF activity (left), but no Ypt31 GEF activity (right). GST and GST–Bet5 complexes were purified from wild-type and trs130ts-mutant cell lysates as described in Fig. 1a. Equal amounts of GST–Bet5-associated complexes from wild-type and mutant cells, as determined in Fig. 1a (1 g), were used in a GDP-release assay for Ypt1 (left) or Ypt31 (right). (b) GST–Bet5-associated complex from trs130 Trs120–Myc mutant cells has increased Ypt1 GEF activity (left), but no Ypt31 GEF activity (right). GST and GST–Bet5 complexes were purified from wild-type and trs130-mutant cell lysates as described in the Fig. 1b legend, and were used in a GDP-release assays as described in a. (c) GST–Bet5-associated complex from trs120 Trs130–Myc mutant cells has increased Ypt1 GEF activity (left), but no Ypt31 GEF activity (right). GST and GST–Bet5 complexes were purified from wild-type and trs120-mutant cell lysates, and were used in GDP-release assays as described in a. Results shown in this figure are the average of duplicate reactions and are representative of 3 independent experiments. Error bars represent s.e.m.

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Trs130 and Trs120 are the two essential TRAPPII subunits⁹, and overexpression of YPT31 suppresses both trs130, and trs120¹². We therefore wanted to determine the role of Trs130 independently of Trs120. The fact that TRAPP purified from trs130-mutant cells contains normal levels of Trs120–Myc (Fig. 1b) indicates that Trs130 itself is necessary for the Ypt31 GEF activity and the lower Ypt1 GEF activity of wild-type TRAPP. To determine the role of Trs120 in TRAPP GEF activity, we used a trs120 strain overexpressing Ypt31, in which Trs130 was tagged with Myc. The level of Trs130 is very low in trs120 cell lysates and, accordingly, there is a very low level of Trs130–Myc in the GST–Bet5-associated complex (Fig. 1c). Thus, in the absence of Trs120, Trs130 is unstable and is not present in the TRAPP complex. TRAPP complexes isolated from trs120-mutant cells show the same Ypt31-defective but Ypt1-elevated GEF activity as TRAPP from trs130-mutant cells (Fig. 2c). The simplest interpretation of these results is that Trs120 is required for the stability of Trs130 and/or its attachment to TRAPP, and therefore for the regulation of the GEF activity of TRAPP on Ypt1 and Ypt31. However, it is possible that, in addition, Trs120 affects TRAPP GEF activity independently of Trs130.

To determine the effect of Trs130 on the GEF activities of TRAPP by an independent method, we separated TRAPPI and TRAPPII, using sizing chromatography. The GST–Bet5-associated complex, purified from wild-type cells expressing Trs130–HA, was applied to a Superdex 200 gel filtration column. Fractions that include complexes with a relative molecular mass >670,000 (M_r >670K) were analysed for the presence of GST–Bet5 and Trs130–HA. As we have previously shown for GST–Bet3 (ref. 5), the GST–Bet5-associated complexes fractionated into two peaks. Immunoblot analysis showed that although both peaks contain GST–Bet5, only the high M_r peak contains Trs130–HA (Fig. 3a). We define the low M_r peak, which does not contain Trs130, as TRAPPI, and the high M_r peak, which does contain Trs130, as TRAPPII (ref. 10). Equal molarities of TRAPPI and TRAPPII, as determined by the level of GST–Bet5, were used in GDP-loss assays with Ypt1 and Ypt31. The TRAPPI fraction specifically acts on Ypt1, whereas TRAPPII specifically acts on Ypt31 (Fig. 3a). This result provides independent support for the requirement of Trs130 for the Ypt31 GEF activity, but not for the Ypt1 GEF activity. It also suggests that Trs130 inhibits the Ypt1 GEF activity of TRAPPII.

Figure 3: TRAPPII-specific subunits regulate Ypt1 and Ypt31 in opposite ways in vitro and in vivo.

(a) TRAPPI and TRAPPII complexes act as specific GEFs for Ypt1 and Ypt31, respectively. TRAPPI and TRAPPII were separated on a gel-filtration column. Purified GST–Bet5-associated complexes (0.5 mg) were applied to a Superdex 200 gel-filtration column. Fractions (500 l) were collected and assayed as follows: upper panel, immunoblot analysis of Trs130–HA (top) and GST–Bet5 or GST (bottom). Sixteen microlitres of column fractions were analysed by SDS-PAGE followed by western blotting using anti-HA and anti-GST antibodies. TRAPPI and TRAPPII are defined by the absence or presence of Trs130–HA protein, respectively. Lower panel, stimulation of GDP release from Ypt1 or Ypt31 by TRAPPI and TRAPPII complexes. Equal amounts of GST- or GST–Bet5-associated complexes (0.45 g), as determined in a, were assayed in 50 l reactions using Ypt1 or Ypt31 as a substrate; 15 min time points are shown. Results shown in this panel are the average of two independent assays and are representative of at least four experiments from two columns. Error bars represent s.e.m. (b) Trs130 regulates the intracellular distribution of Ypt1 and Ypt31/32 in opposite ways. Top: Ypt31 staining is diffuse in trs130ts-mutant cells. Wild-type (VSY459) or trs130ts-mutant (VSY446) cells grown at 26 °C (left), or shifted to 37 °C for 1.5 h (right). Cells were fixed and processed for immunofluorescence (IF) microscopy using affinity-purified anti-Ypt31/32 antibodies. Bottom: Ypt1 staining is enhanced in trs130ts-mutant cells. Cells were grown, fixed and processed as described above, except that affinity-purified anti-Ypt1 antibodies were used for IF microscopy. For each panel, IF is shown at the top, and differential interference contrast (DIC), for cell contour, is at the bottom. Results shown in this panel are representative of at least three independent experiments.

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Opposite effects of the trs130ts mutation on the cellular distribution of the two Golgi GTPases reinforce the opposite effects of Trs130 on Ypt1 and Ypt31 GEF activities. Using immunofluorescence and live-cell microscopy, we examined the affect of trs130ts on the localization of Ypt1 and Ypt31/32. Both Ypt1 and Ypt31/32 show puncate distribution in wild-type cells (Fig. 3b and see Supplementary Information, Figs S2, S3), and these puncta represent the yeast Golgi^{16, 17}. In trs130ts-mutant cells, Ypt31 distribution is more diffuse even at permissive temperature (26 °C), and becomes completely diffuse at the restrictive temperature of this mutant (37 °C; Fig. 3b and see Supplementary Information, Fig. S3). In contrast, the Ypt1 puncta were enhanced in trs130ts-mutant cells at the restrictive temperature of this mutant (100% of cells at 37 °C; Fig. 3b).

The effect on localization is specific for trs130ts and the Golgi Ypts Ypt31/32 and Ypt1, based on the following evidence: first, although the sec7-1ts mutation confers a similar Golgi morphology as trs130ts¹⁸, it does not affect Ypt31/32 and Ypt1 in the same way (see Supplementary Information, Fig. S2a). Second, deletion of RIC1, which encodes a subunit of Ypt6 GEF¹⁹ that functions in endosome-to-Golgi transport^{19, 20} (a transport step that is also regulated by Ypt31/32; ref. 17), does not affect Ypt31/32 cellular distribution (see Supplementary Information, Fig. S2b). Third, the distribution pattern of Sec7 seems normal in trs130ts-mutant cells (see Supplementary Information, Fig. S3), indicating that the Golgi itself, and Golgi localization of another membrane-associated protein, Sec7, are not defective in these cells. Together, these results suggest that Trs130 is required for the proper intracellular localization of Ypt1 and Ypt31/32, and provide in vivo support for the opposite regulatory role of Trs130 on the Golgi Ypts.

As Trs130 is required for the GEF activity and Golgi localization of Ypt31, we wanted to determine whether the two proteins interact physically. Trs130 and the wild-type and the various nucleotide-bound forms of Ypt31 or Ypt1 were cloned into yeast two-hybrid vectors. The expression of the fusion proteins was confirmed by western blot analysis. We found that Trs130 interacted with the nucleotide-free forms of Ypt31, but not with the wild-type, GTP- or GDP-bound forms. In contrast, Trs130 did not interact with any forms of Ypt1 (Fig. 4a). GEFs are expected to interact best with the nucleotide-free form of GTPases, and these forms were shown to act in a dominant-negative manner due to sequestration of the GEF²¹. This result suggests that Ypt31, but not Ypt1, interacts physically with Trs130.

Figure 4: Physical and genetic interactions between TRAPP subunits and the Golgi Ypts.

(a) The nucleotide-free form of Ypt31, but not Ypt1, interacts with Trs130 in the yeast two-hybrid assay. Yeast MATa cells expressing Trs130 from a GAL4-AD (LEU2) vector were mated with MAT cells expressing the various nucleotide-bound forms of Ypt31 or Ypt1 from GAL4-BD (URA3) plasmids: Ypt31 wild type, Q72L (GTP), S27N (GDP), D129N (D-nucleotide-free), N126I (N-nucleotide-free); Ypt1 wild type, Q67L (GTP), S22N (GDP), D124N (D), N121I (N). Growth of diploid cells on SD-Ura-Leu is shown on the top, whereas interaction is shown on the bottom as growth on SD-Ura-Leu-His. Results shown in this panel are representative of three independent experiments. (b) Summary of the interactions between genes encoding the TRAPP subunits Bet3 and Trs130, and the Golgi Ypts Ypt1 and Ypt31. (1) When overexpressed, Ypt31 and Trs130 both suppress each other's growth phenotype. Specifically, overexpression of Ypt31 rescues the growth defect of trs130ts-mutant cells^{11, 12, 13}, and overexpression of Trs130 rescues the growth phenotype of ypt31/32ts-mutant cells, but not of ypt1ts-mutant cells (see Supplementary Information, Fig. S4). (2) Overexpression of Bet3 enhances the growth defect of ypt1ts-, ypt31/32ts- (ref. 5) and trs130ts-mutant cells, but not of sec4ts-mutant cells (see Supplementary Information, Fig. S5). (3) Co-overexpression of Ypt1 with Ypt31, but not with Sec4, has a synergistic positive effect on the growth phenotype of bet3ts, a mutant of a TRAPPI/II subunit (see Supplementary Information Fig. S6). Arrows depict suppression of mutant phenotype (which facilitates growth), whereas flat arrows represent enhancement of the mutant phenotype (which impedes growth). Black indicates interactions that are reported in this study, whereas grey indicates observations from previous studies.

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The physiological relevance of the interaction of the TRAPP subunits with the Golgi Ypts was tested using overexpression analysis. In this approach, a genetic interaction is established if overexpression of one gene either suppresses or enhances the mutant phenotype of the other gene. The observed interactions between the Golgi Ypts, Ypt1 and Ypt31 with the TRAPP subunits Bet3 and Trs130 (from this and previous studies) are summarized in Fig. 4b. Two genetic interactions suggest that Trs130 and Ypt31/32 work together and support the role of Trs130 as a positive regulator of Ypt31. First, overexpression of either YPT31 or TRS130 suppresses the growth phenotype of a mutation in the other. This effect is specific to YPT31 and TRS130, as YPT1 and TRS130 do not interact with each other in a similar way (Refs 11–13, and see Supplementary Information, Fig. S4). Second, if Trs130 is a positive regulator of Ypt31, then TRS130 and YPT31 should interact with BET3 in a similar way. Indeed, overexpression of BET3 has a similar effect on ypt31/32ts- and trs130ts-mutant cells (ref. 5, and see Supplementary Information, Fig. S5).

In addition, two genetic interactions support a role for TRAPP as a GEF for both Ypt1 and Ypt31/32 in vivo. First, overexpression of the TRAPPI/II subunit BET3 has detrimental effects on both ypt1- and ypt31/32-mutant cells, but not sec4-mutant cells (ref. 5, and see Supplementary Information, Fig. S5). Second, overexpression of YPT1 together with YPT31, but not SEC4, has a synergistic positive effect on the growth phenotype of a bet3ts mutant strain (see Supplementary Information, Fig. S6). This synergistic suppression argues against the claim that TRAPP acts as a GEF only for Ypt1 (which is based on a negative biochemical result), but not for Ypt31/32 (ref. 22). Together with the localization effect, the genetic interactions provide in vivo support for the ideas that TRAPP acts as a GEF for both Ypt1 and Ypt31/32 GTPases, and that Trs130 regulates Ypt1 and Ypt31/32 in opposite ways.

Results presented here provide in vitro and in vivo evidence for our suggestion that a Trs120–Trs130 subcomplex is required for the switch of the GEF activity of TRAPP from Ypt1 to Ypt31. In vitro, purified TRAPPI and Trs130-deficient TRAPP from trs120- or trs130-mutant cells, function as specific GEFs for Ypt1 and not Ypt31, whereas TRAPPII, which contains Trs130, specifically functions as a Ypt31 GEF. The two-hybrid interaction of Trs130 with the nucleotide-free form of Ypt31, but not of Ypt1, further supports a role for Trs130 in the Ypt31 GEF activity of TRAPP. The dependence of the protein level of Trs130 on the presence of Trs120, but not vice versa, suggests that Trs120 is required for the stability and/or attachment of Trs130 to TRAPP, and Trs130 in turn is required for the interaction with Ypt31, and the specificity switch of the GEF activity of TRAPP.

In vivo, the trs130 loss-of-function mutation confers opposite effects on the cellular localization of Ypt1 and Ypt31/32. Whereas Ypt31/32 distribution changes from discrete Golgi puncta to diffuse staining, the Ypt1 puncta become more intense (Fig. 3b). The regulation of Ypt/Rab intracellular localization is not well understood. One possibility is that the localization of Ypt–Rabs, together with their regulators and effectors, to specific membrane domains is determined in a combinatorial way. For Ypt1 and Ypt31/32 localization, a subunit of their GEF is crucial. This result also reinforces the notion that Trs130 differentially regulates Ypt1 and Ypt31/32. Genetic interactions provide further support for the physiological relevance of the role of the dissimilar interaction of TRAPP subunits with the Golgi Ypts, and support a role for TRAPP in the coordinated activation of these Golgi gatekeepers.

Based on our results, we propose the following model: TRAPPI, which resides in the cis-Golgi, acts as a GEF for Ypt1, the GTPase that is required for entry into the cis-Golgi. In the trans-Golgi, the Trs120/Trs130 subcomplex joins TRAPP to switch the specificity of the GEF to act on Ypt31, the GTPase that is required for exit from the Golgi (Fig. 5). One version of this model suggests that TRAPPI and TRAPPII exist as two stable complexes on the cis- and trans-Golgi, respectively (Fig. 5a). However, we favour a dynamic model in which the TRAPPII-specific subunits attach to TRAPPI at the trans-Golgi to switch its GEF specificity from Ypt1 to Ypt31 (Fig. 5b). The dynamic model is in agreement with the Golgi cisternal-maturation hypothesis^{23, 24}. In either case, it is important to identify the TRAPP subunit/s that act as the GEF for Ypt1 and Ypt31/32. It would also be interesting to determine the molecular mechanism by which the addition of TRAPPII-specific subunits affects the Ypt GEF specificity of TRAPP. The switch of the GEF specificity between TRAPPI and TRAPPII, from a Ypt1 to a Ypt31 GEF, might be important for ensuring that only one Ypt/Rab is active in a certain compartment. Such a 'switchable', dual-specificity GEF would connect Golgi entry and exit, by consecutive activation of the Golgi gatekeepers Ypt1 and Ypt31/32.

Figure 5: Models for the specificity-switch of the dual TRAPP GEF by Trs130.

The Ypt1 and Ypt31/32 GTPases are required for entry into and exit from the Golgi, respectively^{6, 7}. We propose that the modular GEF complex TRAPP sequentially activates these GTPases. This could occur via two alternative ways, indicated in a and b. (a) TRAPP exists as two independent complexes: TRAPPI, which resides in the cis-Golgi and acts as a GEF for Ypt1, and TRAPPII, which resides in the trans-Golgi and acts as a GEF for Ypt31/32. The TRAPPII-specific subunits Trs120 and Trs130 are required for the specificity switch of TRAPP's GEF activity from Ypt1 to Ypt31/32. (b) TRAPP changes from TRAPPI to TRAPPII on the Golgi. In the cis-Golgi, TRAPPI acts as a GEF for Ypt1. When TRAPP reaches the trans-Golgi, for example, by cisternal maturation^{23, 24}, the TRAPPII-specific subunits, Trs120/Trs130, join the complex, thereby transforming it into a Ypt31/32 GEF. Sequential activation of the gatekeepers of the Golgi, Ypt1 and Ypt31/32, by the same basic GEF complex allows coordination of entry into and exit from the Golgi. ER, endoplasmic reticulum; PM, plasma membrane

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The components of intracellular trafficking machinery are highly conserved from yeast to man. Accordingly, all the essential subunits of the TRAPP complex are conserved among all eukaryotes (ref. 8, and see Supplementary Information, Fig. S1a). Here, we suggest a role for the yeast Trs120 and Trs130 proteins in the specificity switch of the dual-Ypt GEF TRAPP. In contrast, the human homologues of Trs120 and Trs130, NIBP and EHOC-1, were suggested to function in cytokine-induced signalling and as a membrane channel, respectively^{25, 26}. The discrepancy between the suggested functions of the yeast and human proteins will have to be addressed. The conservation of the transport machinery in general, and, although low, of the TRAPP subunits Trs120 and Trs130 specifically, suggests that these proteins act in a similar way in all eukaryotes. We propose that the novel paradigm suggested here, that Ypts are coordinately activated, also applies to their mammalian homologues, Rabs. Importantly, the genes encoding both NIBP and EHOC-1 have been implicated in human disorders^{25, 26, 27, 28}. Elucidation of the mechanism by which TRAPPII-specific subunits function may be relevant for understanding the basis of the human disorders in which NIBP and EHOC-1 have been implicated.

Acknowledgements

We thank D. Stone for critical reading of the manuscript, A. Lodhi for tehcnical help and Y. Jigami for the 2 Trs130 plasmid. This research was supported by grant GM-45444 from the National Institutes of Health (NIH) to N. Segev.

Author Contributions

N.M., Y.L. and A.A.T. were responsible for the experimental work. S.H.C., J.A., Z.L., V.A.S. and S.D.E. contributed plasmids and strains. R.C. performed sequence analysis. N.S., with the help of the other authors, was involved in project planning, data analysis and writing.

Competing interests statement

The authors declare no competing financial interests.

Received 16 June 2006; Accepted 2 August 2006; Published online 15 October 2006.

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