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Vol. 18, Issue 7, 2533-2541, July 2007
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*Department of Biological Sciences, Laboratory for Molecular Biology, University of Illinois at Chicago, IL 60607; and Cell Sciences Imaging Facility, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305
Submitted March 8, 2007;
Revised April 17, 2007;
Accepted April 20, 2007
Monitoring Editor: Vivek Malhotra
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a).
In yeast, Ypt1 and Ypt31/32 regulate transport into and out of the Golgi, respectively (Segev et al., 1988; Jedd et al., 1997). TRAPP is a conserved multisubunit complex that comes in two configurations: TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas TRAPP II functions in late Golgi (Sacher et al., 1998, 2001). We have identified the TRAPP I and TRAPP II complexes as GEFs for Ypt1 and Ypt31/32, respectively (Jones et al., 2000; Morozova et al., 2006). The two TRAPP complexes share seven subunits, and TRAPP II contains three additional subunits, Trs120, Trs130, and Trs65 (Sacher et al., 2000, 2001). Although the two essential TRAPP II-specific subunits, Trs120 and Trs130, are conserved from yeast to humans, the third nonessential subunit, Trs65, is conserved only among some fungi (Cox et al., 2007). Recently, we showed that Trs120 and Trs130 are required for the specificity switch of TRAPP from Ypt1 GEF to Ypt31 GEF (Morozova et al., 2006). However, the role of Trs65 in the GEF function of TRAPP II is not yet clear.
The documented defect of TRS65/KRE11 loss-of-function is in cell wall biogenesis (Brown et al., 1993), and here we point to an additional role for Trs65 in stress response. As for a role in intracellular trafficking, although Trs65 coprecipitates with the TRAPP complex (Sacher et al., 2001; Gavin et al., 2002), its functional connection with this complex until now has been limited to one genetic interaction with a TRAPP I subunit. Specifically, deletion of two nonessential TRAPP subunits, Trs33 (TRAPP I/II) and Trs65 (TRAPP II), results in a synthetic lethal growth phenotype (Tong et al., 2004), and this double mutant can be rescued by overexpression of Ypt31 (Sciorra et al., 2005). This genetic interaction supports a role for Trs65 with the TRAPP complex.
Here, we provide genetic, biochemical, and cellular evidence for the role of Trs65 in the Ypt31/32 GEF activity of TRAPP II. Specifically, Trs65 localizes to the trans-Golgi, interacts genetically and physically with Trs120 and Trs130, and affects TRAPP II function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), affinity-purified rabbit anti-Ypt1 (Segev et al., 1988), rabbit anti-glucose-6-phosphate dehydrogenase (G-6-PDH) (A-9521; Sigma-Aldrich, St. Louis, MO), rabbit anti-glutathione S-transferase (GST) (immunoglobulin [Ig]G fraction; Invitrogen, Carlsbad, CA), horseradish peroxidase-linked anti-rabbit and anti-mouse IgG (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom), and Texas Red dye-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). All chemical reagents were purchased from Sigma-Aldrich, unless otherwise noted.
Culture Conditions
Yeast cells were grown in rich (YPD) media, or minimal (SC) media, supplemented with the appropriate auxotrophic requirements (Rose et al., 1988). Carbon sources were added to 2% (wt/vol). Yeast cells expressing GST-Bet5 or GST under the CUP1 promoter were induced with 0.5 mM CuSO4 for 2 h at 26°C, unless otherwise noted. Yeast transformations were performed by the overnight lithium acetate method (Gietz et al., 1992).
Preparation of Cell Lysates and Protein Analyses
Yeast cell extracts were prepared as described previously (Chen et al., 2005). Cell breakage buffers were supplemented with an EDTA-free protease-inhibitors cocktail (Roche Diagnostics). Protein concentrations were determined by a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Ten micrograms of yeast whole cell lysates were loaded on 10% SDS-polyacrylamide gel electrophoresis (PAGE). Gels were run, and proteins were transferred to polyvinylidene difluoride membranes and subjected to immunoblot analysis. Quantification of protein bands was done using the AlphaEase FC and Alpha-Imager (Alpha Innotech, San Leonardo, CA).
Purification of GST Fusion Proteins
Ypt1 and Ypt31 proteins expressed in bacteria were purified as described previously (Jones et al., 1995). GST-tagged proteins expressed in yeast were purified as described previously (Morozova et al., 2006). The total protein concentration of the eluted fractions ranged between 0.05 and 0.4 mg/ml. GST-associated complexes (0.2 µg) were tested by immunoblot analysis as described above for lysates.
GDP Release Assays
GDP release assays were performed as described previously (Morozova et al., 2006). GST–Bet5- or GST-associated complexes were purified from yeast and added to the reaction as a source of GEF.
Fluorescence Microscopy
Immunofluorescence microscopy was performed as described previously, using affinity-purified anti-Ypt1 and anti-Ypt31/32 antibodies (Jedd et al., 1997). TRS65 was tagged on the chromosome with yellow fluorescent protein (YFP) at the N terminus in wild-type (BY4741/NSY825) and Sec7-DsRed (NSY986; Chen et al., 2005) strains as described previously (Prein et al., 2000) to provide NSY1179 and NSY1180, respectively. Briefly, YFP was amplified by polymerase chain reaction (PCR) from pDH22 (pNS610) and then transformed into yeast cells. Correct targeting was verified by diagnostic PCR and PCR-product sequencing. The strain expressing both YFP-Trs65 and Cop1-red fluorescent protein (RFP) was constructed by mating and dissection of NSY1179 with NSY862. DsRed-FYVE plasmid (pNS716) was transformed into NSY1178 for testing the colocalization of YFP-TRS65 with this endosomal marker. Cells expressing Sec7-DsRed and Cop1-RFP were in YPD to mid-log phase, whereas cells expressing DsRed-FYVE were grown in SD-Leu to mid-log phase and then switched to SD-Leu-Met for 2 h. Live cell deconvolution microscopy was performed as described previously (Chen et al., 2005). Briefly, a series of 5–10 Z stacks, 275 nm each, were collected for each field using a 63x objective, and deconvolved using regularized inverse filter and Axiovision 4.3 software (Carl Zeiss, Thornwood, NY).
General Secretion Assay
Yeast cells were grown at 26°C to log phase. Ten OD6oo units of cells were spun at 3000 rpm for 5 min. The cell pellet was washed twice with 5 ml of SD-Cys-Met, and then it was resuspended in 1 ml of this medium. Cells were preincubated at 37°C for 20 min and then pulsed with 10 µCi/OD Trans35S label for 1.5 h at 37°C. Media proteins were analyzed after trichloroacetic acid (TCA) precipitation as described previously (Gaynor and Emr, 1997).
Electron Microscopy
Yeast cells were grown at 26°C to early log phase (0.5 OD600). Half the culture was left at 26°C, and the other half was shifted to 37°C for 1.5 h. Cells were fixed in 0.2 M sodium cacodylate buffer, pH 6.7, and 4% glutaraldehyde (both reagents are from Ted Pella, Redding, CA), and they were processed for electron microscopy as described previously (Byers and Goetsch, 1975).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To determine whether the intracellular localization of Trs65 is similar to that of TRAPP II, we compared the localization pattern of Trs65 to that of Trs130. The colocalization of Trs130-GFP with the late Golgi marker Sec7, and not with the endosomal marker FYVE domain, was determined previously by direct fluorescence microscopy (Cai et al., 2005). Trs65 was tagged on the chromosome at its N terminus with YFP. YFP-Trs65 is functional because it does not confer a cell wall defect, similar to that displayed by trs65
mutant cells (Supplemental Figure S1). The intracellular localization of YFP-Trs65 was determined by its colocalization with known compartmental markers labeled with a red fluorescent tag, by using live cell deconvolution microscopy. Figure 1 demonstrates that Trs65 shows strong colocalization with the trans-Golgi marker Sec7 and partial colocalization with the early Golgi marker Cop1. In addition, as has been reported previously for Trs130, Trs65 does not colocalize with the endosomal marker FYVE domain. In summary, Trs65, like another TRAPP II GEF subunit, Trs130, and the substrate of this GEF, Ypt31 GTPase (Chen et al., 2005), localizes primarily to the trans-Golgi. These results indicate that Trs65 not only coprecipitates with TRAPP II but also colocalizes with it mainly to the trans-Golgi.
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(ts). The temperature sensitivity of the triple mutant can be complemented by GST-tagged Trs65 expressed from a 2µ plasmid (Figure 2B). This synthetic interaction suggests that the tagged versions of the Trs120 and Trs130 are somewhat impaired for function and that Trs65 functions together with Trs120 and Trs130.
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cells has been in cell wall biogenesis (Brown et al., 1993). We used the trs65(ts) mutant cells to search for protein-trafficking defects. First, we tested the ability of trs65(ts) mutant cells to secrete Hsp150 to the medium (Gaynor and Emr, 1997). At the nonpermissive temperature, trs65(ts) mutant cells exhibit a reduction in secretion of Hsp150. This defect is less severe than the secretory defect of trs130ts mutant cells in the same assay, but it was significant (Figure 2C). Second, mutants defective in exit from the Golgi, such as sec7, trs130, and ypt31/32, exhibit accumulation of abnormally large Golgi, termed Berkeley bodies (Novick et al., 1981; Jedd et al., 1997; Sacher et al., 2001). The trs65(ts) mutant cells, at their nonpermissive temperature, show accumulation of Berkeley bodies (Figure 2D). Together, these results place Trs65 with the other two TRAPP II-specific subunits Trs120 and Trs130, and they suggest that it functions in the exit from the trans-Golgi.
If Trs65 functions in TRAPP II together with Trs120 and Trs130, we expect that trs65(ts) would exhibit genetic interactions similar to those conferred by the trs120 and trs130 loss-of-function mutations. First, we tested the trs65(ts) triple mutation for interaction with genes encoding the TRAPP I/II subunits Bet3 and Bet5. We have reported previously that overexpression of these two subunits, which are shared by TRAPP I and TRAPP II, enhance the growth phenotype of trs130 and trs130ts mutant cells (Morozova et al., 2006). Here, we show that overexpression of Bet3, but not Bet5, enhances the growth phenotype of trs120 mutant cells (Figure 3A). We also show that overexpression of Bet3 and Bet5 enhances the growth phenotype of trs65(ts) mutant cells (Figure 3B). Second, it has been shown previously that overexpression of Ypt31/32, but not Ypt1, rescues the growth phenotype of trs120, trs130, and trs130ts mutant cells (Yamamoto and Jigami, 2002; Zhang et al., 2002; Sciorra et al., 2005). Here, we show a similar genetic interaction with the trs65(ts) triple mutation. Specifically, overexpression of Ypt31, but not Ypt1, rescues the growth defect of trs65(ts) mutant cells at 37°C (Figure 3C). Together, the similarity of genetic interactions of the trs65(ts) triple mutation to those of trs120 and trs130 (summarized in Figure 3D) further supports a common role for these TRAPP II-specific subunits.
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; Gavin et al., 2002). Here, we used the yeast two-hybrid assay to test for physical interaction of Trs65 with the TRAPP II-specific subunits Trs120 and Trs130. Cells expressing Trs65 fusion with a binding domain were mated with cells expressing Trs120 or Trs130 fusion with an activation-domain. Growth on plates without histidine shows interaction of Trs65 with both Trs120 and Trs130 (Figure 4). These interactions are also detectable on plates without histidine in the presence of 5 mM 3-amino-1,2,4-triazole and under the most restrictive selection for interaction: media without adenine. We know that these interactions are specific, because controls using empty vectors and the TRAPP I/II subunit Trs23 in this yeast two-hybrid assay do not show any growth on the selective medium. Similar negative results were obtained with three other TRAPP I/II subunits: Bet3, Bet5, and Trs31 (data not shown). These negative results indicate that the physical interaction of Trs65 with the other two TRAPP II-specific subunits in the yeast two-hybrid assay is not due to an indirect interaction with the whole TRAPPI/II complex, as is the case with the affinity-capture interactions.
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). Because we found here that Trs65 interacts with these two TRAPP II-specific subunits genetically and physically (see above), we wanted to determine whether Trs65 affects TRAPP II complex composition. The protein levels of the tagged versions of two essential TRAPP II-specific subunits, Trs120 and Trs130, in trs65 cell lysates were determined by immunoblot analysis and compared with those present in wild-type cell lysates. We found that the level of Trs130-HA protein in trs65 cell lysates is lower than its level in wild-type lysates (
50% at 37°C). The level of Trs130-HA, but not Trs120-myc, is also lower in trs65(ts) triple mutant cell lysates even at 26°C, and this effect is more severe at 37°C (Figure 5A). These results show that Trs65 is important for the normal cellular level of the Trs130 protein.
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mutant cells expressing only Trs130-HA, there was a lower level of the tagged Trs130 than in TRAPP complexes purified from wild-type cells. Lysates and TRAPP complexes purified from trs65(ts) mutant cells overexpressing GTS-Bet5 have lower levels of both tagged Trs130 and Trs120, when compared with wild-type lysates and TRAPP complexes (Figure 5). This effect could be due to the negative synthetic growth defect of overexpressing Bet5 in trs65(ts) mutant cells described above (Figure 3B). Together, these results show that deletion of TRS65 confers in a lower cellular level of Trs130, as well as a lower Trs130 level in TRAPP complexes.
Loss of Trs65 Affects the Ypt GEF Activity of TRAPP
We have previously shown that loss of Trs120 and Trs130 function results in reduced Ypt31/32 GEF activity of TRAPP. In addition, TRAPP complexes purified from trs120 or trs130 mutant cells possess higher Ypt1 GEF activity (Morozova et al., 2006). Because TRAPP complexes purified from trs65 mutant cells have low levels of Trs130, we expected that these complexes would be defective in their Ypt GEF activity. To test this possibility, TRAPP complexes purified from trs65 mutant cells were tested for stimulation of GDP release from Ypt31 and Ypt1 proteins. GST–Bet5-purified complexes from trs65 mutant cells, in which neither Trs130 nor Trs120 were tagged, have lower Ypt31 GEF activity compared with wild-type complexes. However, the Ypt1 GEF activity remained unaffected (Figure 6A). Moreover, complexes purified from trs65 mutant cells in which only Trs130 is tagged show the same result (Figure 6B), suggesting that tagging Trs130 alone in trs65 mutant cells does not affect the Ypt1 GEF function of TRAPP. Finally, TRAPP complexes purified from trs65(ts) mutant cells (in which both Trs120 and Trs130 are tagged) grown at their permissive temperature (26°C) also showed similar results. Only when the trs65(ts) mutant cells were grown at their nonpermissive temperature (37°C), the Ypt31 GEF activity of TRAPP was abolished, and the Ypt1 GEF activity was higher than that of wild-type TRAPP (Figure 6C). In experiments using Ypt32 as a substrate, we got results similar to those of Ypt31 (data not shown). Thus, when the level of Trs130 is reduced in trs65 mutant cells, only the Ypt31/32 GEF activity is affected. However, when the level of all three TRAPP II-specific subunits is reduced, the GEF specificity switch of TRAPP from Ypt1 to Ypt31/32 is also affected. These results suggest that Trs65 contributes to the Ypt GEF activity of TRAPP, probably through its effect on the TRAPP II complex assembly or stability.
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). To determine the effect of the trs65 mutation on the localization of the Golgi Ypts, the localization of Ypt1 and Ypt31/32 in wild-type and mutant cells was compared by immunofluorescence microscopy. In trs65 mutant cells in which Trs120 and Trs130 are not tagged, the localization of Ypt31 is more diffuse than in wild-type cells, especially at 37°C, but there is no effect on the Ypt1 punctate staining pattern in these cells (Figure 7A). In trs65(ts) mutant cells at the nonpermissive temperature, the localization of both Ypt31 and Ypt1 are affected in opposite ways: the Ypt31/32 localization is diffuse, whereas the Ypt1 staining is brighter (Figure 7B). Thus, the Ypt localization results are in agreement with those of the Ypt GEF activity: the trs65 mutation, which affects only the Ypt31/32 GEF activity, affects only the localization of this GTPase pair. In contrast, the more severe trs65(ts) triple mutation, which affects the GEF activities for Ypt1 and Ypt31/32 in opposite ways, also affects the localization of these GTPases in opposite ways. Membrane attachment of Ypt1 and Ypt31/32 in trs130 mutant cells, as determined by cell fractionation analysis, was found to be similar to that of wild-type cells (our unpublished data). We therefore expect that trs65 will also not have an effect on the Ypts distribution between membrane and cytoplasm. We suggest that TRAPP II is not required for membrane attachment of the Ypts, but it is required for their proper localization to the right cellular compartment.
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; Gavin et al., 2002) and functional analysis presented here suggest a role for Trs65 in intracellular trafficking. However, TRS65/KRE11 was first identified as a gene important for cell wall biogenesis based on its mutant phenotype (Brown et al., 1993). We wanted to determine whether Trs65 is a multifunctional protein and compare it with the other nonessential TRAPP subunits. For that, we analyzed the Saccharomyces Genome Database (SGD; Ball et al., 2000; as of February 25, 2007) for all the genetic and physical interactions as well as mutant phenotypes reported for TRS65/KRE11, including those obtained from genome-wide studies. Genetic and physical interactions detailed in BioGrid (Stark et al., 2006) support a role for Trs65 in three different cellular processes: cell wall biogenesis (only genetic interactions), stress response, and membrane trafficking (summarized in Supplemental Table S2). Mutant phenotypes support a role for Trs65 in two of these processes: cell wall biogenesis (Brown et al., 1993) and stress response (Supplemental Table S3). The latter is suggested by the sensitivity of trs65 mutant cells to three agents that induce oxidative stress: dithiothreitol, diamide, and paraquit (Prophecy; Fernandez-Ricaud et al., 2007). Here, we demonstrate that trs65 mutant cells exhibit a phenotype also in the third cellular process: TRAPP II function in intracellular trafficking. Together, the interactions and mutant phenotypes show that Trs65/Kre11 plays a role in three different cellular processes (Figure 8).
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(Supplemental Figure S1 and Table 2). However, the interaction of Trs33 and Trs85 with cell wall biogenesis regulators is limited to one case: both have a weak genetic interaction with GAS1 (synthetic growth defect, as opposed to the synthetic lethality exhibited with TRS65). For stress response, deletion of TRS33, but not TRS85, results in sensitivity to two of the three agents that induce oxidative stress response (Table 2). However, only Trs85, but not Trs33, shows one physical interaction with one protein that plays a role in stress response (Table 1 and Supplemental Tables S3–S5). Thus, only Trs65 shows both mutant phenotypes and multiple genetic and physical interactions that connect it with cell wall biogenesis and stress response. This survey suggests that not all regulators of protein trafficking play an equal role in the two other cellular processes for which Trs65 is important.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and genetic interactions reviewed here support this role (Supplemental Table S2). A role for Trs65 in stress response is suggested here based on mutant phenotypes and genetic and physical interactions (Figure 8, Tables 1 and 2, and Supplemental Tables S2 and S3). Last, a role for Trs65 in protein trafficking has been suggested previously, based on its physical interaction with the TRAPP complex and its genetic interaction with the nonessential TRAPP I/II subunit Trs33 (Tong et al., 2004; Sciorra et al., 2005). Here, we provide the first functional evidence that connects Trs65 to the TRAPP II complex.
The idea that Trs65 functions as part of the TRAPP II complex is based on the following cellular, genetic, and biochemical evidence. First, Trs65 localizes to the Golgi, like Trs130 and Ypt31. Second, TRS65 interacts genetically with TRS120 and TRS130 to yield the trs65(ts) triple mutation. At their nonpermissive temperature, trs65(ts) mutant cells confer a partial defect in the secretion of Hsp150 to the medium, accumulate aberrant Golgi structures similar to those accumulated in trs130ts and ypt31/32ts mutant cells (Jedd et al., 1997; Sacher et al., 2001), and exhibit genetic interactions similar to those of trs120 and trs130 mutant cells. Third, Trs65 interacts physically with both Trs120 and Trs130 in the yeast two-hybrid assay. Last, loss of Trs65 function results in a lower level of Trs130 in cell lysates and in TRAPP complexes. The level of both Trs120 and Trs130 is reduced in TRAPP complexes purified from trs65(ts) mutant cells overexpressing GST-Bet5. Loss of Trs65 function also affects the Ypt GEF activity of TRAPP and the localization of the Golgi Ypts.
Recently, we suggested a role for Trs120 and Trs130 in switching the GEF specificity of TRAPP II from Ypt1 to Ypt31/32. This idea was based on the findings that trs120 and trs130 mutations result in opposite effects on the GEF activity and localization of Ypt1 and Ypt31/32 (Morozova et al., 2006). We also suggested that Trs120 is required for the stability of Trs130 or to its attachment to the TRAPP complex, based on the finding that the protein level of Trs130 depends on Trs120, but not vice versa (Morozova et al., 2006). The simplest explanation for results presented here is that Trs65 also contributes to the interaction of Trs130 with the TRAPP complex and that uncomplexed TRAPP II-specific subunits are more susceptible to degradation. Furthermore, Trs65 exerts its effect on the Ypt GEF activity of TRAPP II and the localization of the Golgi Ypts through its interactions with the other two essential subunits of this complex, Trs120 and Trs130. In summary, we propose that Trs65 contributes to the assembly and/or stability of TRAPP II and thereby to the GEF activity of this complex (Figure 9).
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effect on Trs130 and Trs120 levels, and the effect on GEF activity and localization of Ypts. In trs65 mutant cells, when only Trs130 level is reduced, only the Ypt31/32 GEF activity and localization is affected. In trs65(ts) mutant cells, when the level of both Trs120 and Trs130 is reduced, Ypt1 GEF activity and localization are affected as well. This idea also explains the genetic interaction between trs65 and the tagged TRS120 and TRS130. If Trs65 helps stabilizing and/or localizing Trs120 and Trs130 to TRAPP II, and the interactions of tagged Trs120 and Trs130 are somewhat compromised, this might result in increased dependency on Trs65 for their stability in trs65(ts) triple mutant cells.
The mechanisms and regulation of intracellular trafficking have been extensively studied in the last decade. In contrast, our understanding of the coordination of this process with other cellular processes is still vague. In Saccharomyces cerevisiae, Trs65 is important for three different cellular processes; therefore, it is a candidate for their coordination. One possibility is that the three processes in which Trs65 plays a role are dependent on each other, e.g., cell wall biogenesis is dependent on intracellular trafficking (Ortiz and Novick, 2006), and the stress response is dependent on the integrity of the cell wall (Valdivia and Schekman, 2003). In such a case, we expect all intracellular trafficking regulators to have connections with the other two processes similar to those of Trs65. However, our survey of the two other nonessential TRAPP subunits suggests this is not the case (Tables 1 and 2). An alternative possibility is that not all regulators of intracellular trafficking affect the other two cellular processes and that Trs65 is a multifunctional regulator.
Interestingly, our phylogenetic analyses of the TRAPP II- specific subunits showed that unlike the conserved and essential subunits, Trs65 is only present in some fungi (Cox et al., 2007). One explanation for the lack of Trs65 conservation is that higher eukaryotes have a functional homologue for Trs65, which has lost the sequence similarity. Alternatively, Trs120 and Trs130 in higher cells do not need the Trs65 function, because Trs65 serves to connect TRAPP with fungi-specific functions, e.g., cell wall biogenesis. Regardless, it is important to investigate the possible role of Trs65 in coordination of the three cellular processes in which it plays a role: intracellular trafficking, cell wall biogenesis, and stress response.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Nava Segev (nava{at}uic.edu)
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cells expressing Trs65 from pGBDU-C2 (GAL4-BD, URA3) vector. Diploids were selected on SD-Ura-Leu medium. The growth control of the diploids is shown on the left (-Ura-Leu), and interaction is shown at the right (-Ura-Leu-His). Cells were plated in 10-fold serial dilutions from top to bottom and were incubated at 30°C. Empty vector (ø) for both plasmids are shown as negative controls. Results shown in this figure are representative of at least two independent experiments.
