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Originally published In Press as doi:10.1074/jbc.M307446200 on November 10, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4498-4506, February 6, 2004
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Inhibition of Sodium/Proton Exchange by a Rab-GTPase-activating Protein Regulates Endosomal Traffic in Yeast*

Rashid Ali, Christopher L. Brett{ddagger}, Sanchita Mukherjee, and Rajini Rao§

From the Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, July 11, 2003 , and in revised form, November 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The acidification of compartments along the biosynthetic, endocytic, and secretory pathway is critical for their cellular function (1). The low pH environment generated by the vacuolar H+-pumping ATPase is essential for a number of well defined processes, such as the activation of endoproteases and the dissociation of receptor-ligand complexes. In addition to providing an optimal environment for cargo, compartmental pH appears to play a signaling role in controlling vesicular traffic. For example, van Weert et al. (2, 3) showed that intraorganellar acidification was important for late endosome to vacuole trafficking, whereas inhibition of the vacuolar proton pump induces retrograde transport from the trans-Golgi network (TGN)1 to the Golgi. In addition to the vacuolar H+-pumping ATPase, the intraendosomal acidification machinery includes a chloride channel and a Na+/H+ exchanger. The activities of these transporters are predicted to be crucial in regulating compartmental pH, consistent with the recent discovery of specific endosomal isoforms of the CLC and NHE gene families in organisms ranging from yeast to human (4–8). Mutations in the CLCN5 gene lead to Dent's disease, clinically manifested as low molecular weight proteinuria, and provide a link between endosomal acidification and regulation of receptor-mediated endocytosis in the proximal kidney tubule (9). In yeast, disruption of the endosomal Na+/H+ exchanger NHX1 leads to defective vesicular trafficking out of the prevacuolar compartment/late endosome, resulting in enlargement of the PVC and a vacuolar protein sorting phenotype (vps44; 10).

Although it is increasingly apparent that there is a critical requirement for specific luminal ionic concentration and pH in vesicular trafficking, the molecular basis for this requirement is unknown. The existence of a transmembrane pH sensor protein in the endosomes, which communicates the acidity of the lumen to regulatory proteins in the cytosol, has been postulated (10–13). One intriguing regulatory mechanism involves the intraendosomal pH-dependent association of vesicles with a member of the Arf family of small GTPases, its cognate GDP/GTP exchange factor ARNO, and other coat proteins in pancreatic microsomal vesicles and proximal tubule endosomes (11, 14). Thus, although the identity of the hypothetical pH sensor remains to be defined, there are emerging insights into the regulatory role of pH in vesicular trafficking.

In this work, we forge a new link between endosomal pH and trafficking by demonstrating an interaction between the C-terminal tail of yeast Nhx1 and Gyp6, a protein that activates GTP hydrolysis on the Ypt/Rab family of small GTPases (15). There are six Gyp proteins in yeast which share a conserved catalytic GTPase-activating (GAP) domain. Of the 11 Ypt family members in yeast, Ypt6, the homolog of the mammalian Rab6 GTPase, plays a role in retrieval of proteins from the endosome to the Golgi (16). Ypt7 is required for fusion of vesicles derived from endosome with the vacuole, through an interaction with the HOPS complex and Vam7, a homolog of the neural SNAP25 protein (17, 18). Importantly, the preferred substrates of Gyp6 in vitro are Ypt6 and Ypt7 (15). Evidence presented in this work supports a functional coupling between Nhx1-dependent events at the donor compartment and Ypt6/Ypt7-regulated vesicle fusion at the acceptor compartment(s). Our functional data, taken together with the physical interaction between Gyp6 and Nhx1, indicate that Gyp6 is a negative regulator of Na+/H+ exchange activity of Nhx1. Because the GAP activity of Gyp6 serves to terminate the active signal in Ypt6-GTP, Gyp6 can also be considered a negative regulator of Ypt6-mediated vesicle fusion in the PVC to Golgi retrograde pathway. Furthermore, we show that Ypt6 and Nhx1 function along the same pathway. We propose a model in which reciprocal interaction of Nhx1 and Ypt6 with a common negative regulator serves to coordinate anterograde and retrograde traffic in the Golgi to PVC pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Growth Media—Saccharomyces cerevisiae strain PJ64-4A (19) was used for the yeast two-hybrid screen. Deletion mutants of strain BY4741 (MAT a his3D1 leu2D0 met15D0 ura3D0) and BY4742 (MAT {alpha} his3D1 leu2D0 lys2D0 ura3D0) used in this study were purchased from ResGen, Invitrogen. NHX1 deletion in the same background was made essentially as described by Nass et al. (20). All yeast strains were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) or SC medium (minimal medium containing 2% glucose, 0.5% ammonium sulfate, 0.17% yeast nitrogen base without amino acids and without ammonium sulfate; BIO 101, Inc.). Histidine (30 µg/ml), leucine (0.l mg/ml), uracil (30 µg/ml), methionine (0.1 mg/ml), and lysine (0.1 mg/ml) were added when needed. Growth assays of hygromycin B and low pH sensitivity were in APG medium (10 mM arginine, 8 mM phosphoric acid, 2% glucose, 2 mM MgSO4, 1 mM KCl, 0.2 mM CaCl2, and trace minerals and vitamins). Growth assays were performed by inoculating 1 ml of APG medium with overnight cultures grown in selective SC medium. Cells were washed three times with sterile distilled deionized water prior to inoculation. Growth was monitored by measuring A600 nm after culturing for 24 h at 30 °C.

Two-hybrid Screening—A Gal4-based yeast two-hybrid system (19) was used to screen for Nhx1-interacting proteins. The last 119 amino acids from the C-terminal tail of Nhx1 were cloned into the "bait" vector pGAD-C1 (pGAD-NHX1515–633) and used to screen a yeast pGBD-C1 genomic DNA library. 3.5 x 105 Trp+ and Leu+ colonies were screened on quadruple drop-out plates (-Leu, -Trp, -His, and -Ade). Individual colonies containing potentially positive clones were isolated, retested for interaction with Nhx1 on quadruple drop-out plates supplemented with 20 µg/ml X-{alpha}-gal (Clontech). Plasmid pGBD-GYP6271–459, containing the C-terminal region of GYP6, was isolated from a positive clone and transformed into Library Efficiency DH5{alpha}-competent cells (Invitrogen) for amplification, followed by restriction analysis and sequencing.

Recombinant DNA Techniques—The GYP6 gene was cloned by amplification of genomic DNA from S. cerevisiae using PCR and the following primers: sense 5'-CGCGGCACGCGTAAGGATGTCTTACAATGGGCG-3' and antisense 5'-GGCGCGCGGCCGCTTATTGGCTTATTTTCTTTTGTTC-3' containing the NotI and MluI restriction sites, respectively. The GYP6 open reading frame was inserted behind the PGK promoter and N-terminally tagged with GFP, CFP, or His6. Plasmid pUG23-GYP8-CFP was a gift from Prof. Dieter Gallwitz (21). Plasmid harboring NHX1 tagged with either a C-terminal triple HA epitope or GFP tag, under the control of NHX1 endogenous promoter, was described earlier (4). An inverse PCR based strategy was used to generate deletion fragments of the 119-amino acid C-terminal tail of Nhx1 in pGAD-C1 vector (22). Deletions were confirmed by sequencing. Plasmids were then transformed into PJ64-4A yeast strain along with the C-terminal fragment of GYP6 in pGBD-C1 (pGBD-GYP6271–459) and assayed for reporter gene activation as described above.

Extraction of GYP6 from Membranes—Total membrane preparations from yeast strains expressing His::GYP6 and NHX1::HA were prepared as described by Wells and Rao (23) and treated with one of the following: 10 mM potassium phosphate, pH 7.5, 1% Triton X-100, 20 mM EDTA, 300 mM KCl, 600 mM NaCl and 50 mM Na2CO3, on ice for 15 min with occasional vortexing. To pellet the insoluble fraction, the mixture was centrifuged at 65,000 rpm at 4 °C for 30 min in Beckman TLA100.3 rotor. Both soluble (supernatant) and insoluble (pellet) fractions were analyzed by SDS-PAGE and immunoblotting.

Pull-down Assay—Total membrane preparations (1 mg) from yeast expressing His::GYP6 or NHX1::HA or both were solubilized in buffer containing 300 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 20 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma) and incubated overnight with Ni-NTA agarose beads at 4 °C. The beads were then washed four times with wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 20 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma)). Proteins were eluted by incubating Ni-NTA beads with 150 mM imidazole buffer and separated on a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and detected with anti-His (Clontech; 1:8,000 dilution) and anti-HA (Covance; 1:2,000 dilution) monoclonal antibodies.

Fluorescence Microscopy—Yeast cells were grown in selective medium at 30 °C to logarithmic phase. A 1-ml culture was briefly centrifuged to pellet the cells. The cells were then washed twice with 50 mM succinic acid and 2% glucose solution, pH 5.5, and then resuspended in 50 µl of the same solution. Cell suspensions of 4 µl were dropped on poly-L-lysine-treated coverslips and placed on slides. Samples were viewed on a Zeiss Axiovert 200 microscope equipped with an Ultraview confocal scanner from PerkinElmer Life Sciences using a Zeiss 100x oil immersion lens. Digitized images (16-bit) were acquired with a Hamamatsu ORCA-ER camera and Ultraview imaging software (PerkinElmer Life Sciences). FM4-64 staining of the PVC was done in the same solution using a protocol described by Shin et al. (24) which specifically labels this compartment.

CPY Plate Assay—Yeast cultures were freshly grown in SC medium (A600 = 4) and washed with deionized and distilled water. 5-µl aliquots of the dilutions indicated in figure legends were spotted on a nitrocellulose membrane (Amersham Biosciences) which was placed on the surface of YPD or SC plates and incubated at 30 °C for 12–18 h. The membranes were lifted and washed with deionized and distilled water to remove all cells. Proteins adsorbed on the membrane were detected by immunoblotting using monoclonal CPY antibody (Molecular Probes; 1:2,000 dilution).

Vacuolar pH Measurements—To measure vacuolar pH, seed cultures were grown and washed as described above and then used to inoculate 0.2-ml cultures within a clear bottomed, black 96-well microtiter plate. Growth was monitored by measuring A600 nm after culturing for 18 h at 30 °C in specified growth medium (APG, pH 4.0 and 2.7). Cells were then collected by centrifugation, resuspended in the same specified growth medium containing 50 µM BCECF-acetoxymethyl ester, and incubated at 30 °C for 20–30 min (see Ref. 25). Cells were collected again by centrifugation, washed three times, resuspended in 200 µl of APG growth medium at the indicated pH, and immediately used for fluorescence measurement.

Fluorescent intensity and absorbance values were acquired using a BMG FLUOstar Optima multimode plate reader with accompanying BMG FLUOstar Optima Version 1.20-0 software (BMG Labtechnologies, Durham, NC). Single emission fluorescence intensity readings at 450 nm (I450) and 490 nm (I490) excitation wavelengths and A600 nm readings were acquired, and background fluorescence was measured using BCECF-free cultures. At the end of each experiment, a calibration curve of fluorescence intensity versus pH was obtained for each yeast strain tested by incubating yeast cultures in 200 µl of experimental medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3,10 mM 2-deoxyglucose, 50 µM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values within the range of 4.0 to 8.0 using 1 M NaOH (25). However, given that the pKa value of BCECF is near 7.2 (data not shown), the I490 /I450 could not be used to estimate accurately pH values below 5.0, which is the case for vacuolar pH when yeast are gown in low pH media. Thus, as an alternative, background-subtracted I490 values normalized to cell density (NI490) were plotted versus pH (Fig. 1). The figure shows that NI490 values decrease as pH decreases within a pH range of 5.0–4.0. To estimate vacuolar pH, experimental NI490 values from a given strain were compared with a calibration curve generated from the same yeast strain, as growth and the amount of dye loaded varied between strains (data not shown). Data are reported as means ± S.E., and statistical comparisons were performed with Student's two-tailed t tests (paired or unpaired, as appropriate); significance was assumed at the 5% level.



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  		FIG. 1.
BCECF is an indicator of yeast vacuolar pH. BCECF-loaded yeast were incubated in calibration buffer titrated to pH 4.0, 5.0, 6.0, 7.0, or 8.0 as described under "Materials and Methods." Fluorescence intensity values (arbitrary units) were recorded at 490 nm and normalized to cell number (NI490; data are from three to five independent experiments for each of seven strains tested). Each symbol represents mean data from a different strain shown in the inset; a nonlinear curve fit of all data is shown as a dark line. Inset, fluorescent micrographs of BCECF-loaded yeast indicating compartmentalization of the dye (blue) in all seven strains examined.

 
Measurement of [14C]Methylammonium Accumulation—Cells were grown in SC with the required supplements to an absorbance at 600 nm of 0.4–0.5, washed twice to remove potassium, and resuspended at the same concentration in a potassium starvation medium containing 2% glucose, 50 mM succinic acid (adjusted to pH 5.5 with Tris), and 1% casein hydrolysate (Sigma). After a 4-h incubation, the cells were centrifuged, washed, resuspended in water, and stored on ice. For uptake measurements, the cells were diluted to O.D. 10 in medium containing 4% glucose, 1% casein hydrolysate, and 50 mM succinic acid adjusted to pH 5.5 with Tris. After a 5-min preincubation, [14C]methylamine hydrochloride (0.2 mM and 2.5 µCi/ml final concentration; ICN) was added from concentrated stock solutions. After 40 min of incubation, the transport reaction was stopped by diluting samples of 100 µl with 10 ml of ice-cold 20 mM MgCl2, and cells were collected by vacuum filtration through a 0.45-µm pore size nitrocellulose filter (Millipore HAWP) and washed three times on the filter with 20 mM ice-cold MgCl2. Moist filters were dissolved with 1 ml of N,N,-dimethyl formamide, then 10 ml of scintillation mixture was added (Cytoscint from ICN), and radioactivity was monitored using a Beckman LSA 1801 liquid scintillation counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physical Interaction of Nhx1 with Gyp6
The N-terminal transporter domain of the NHE family of Na+/H+ exchangers contains 12 well conserved membrane-spanning segments followed by a more variable C-terminal cytoplasmic domain of 150–350 amino acids (26). In representative members of the plasma membrane subfamily of NHE, regulation of exchanger activity by growth hormones, Ca2+/calmodulin, cytoskeletal attachment, and other signaling cascades is well established and is mediated through numerous protein-protein interactions via the C-terminal domain (27–29). Similar information on the newly recognized intracellular subfamily of NHE, which include yeast Nhx1 and human NHE6 and NHE7 (6, 7), is lacking. To find proteins interacting with Nhx1, we performed a yeast two-hybrid screen using the C-terminal 119 amino acids of Nhx1 cloned into plasmid pGBD-C1 as bait, in conjunction with the yeast genomic library YL2H-C1 (19). A total of 3.4 x 105 colonies were screened on quadruple selection (Trp-, Leu-, Ade-, and His-) media, from which several positive clones were obtained. One of these was identified as the C-terminal 188-amino acid fragment of Gyp6, a GAP for the Rab family GTPase, Ypt6. The Nhx1 interacting region of Gyp6, from residue 271 to 459, lies mostly outside the conserved catalytic domain of the Gyp family, which extends from residues 33 to 310 (30). Because the long C-terminal tails of mammalian NHE have discrete subdomains that have been associated with binding to individual physiological partners, we sought to identify the minimal stretch of amino acid sequence in Nhx1 required for interaction with Gyp6. Fig. 2 shows the results from the two-hybrid assay between GYP6271–459 and a set of deletions in the Nhx1 tail domain. The C-terminal 64 amino acids (row f), but not the N-terminal 55 amino acids (row e), of the Nhx1 tail were capable of activating reporter genes in the two-hybrid assay, although the strength of the signal was somewhat less than that of the original 119-amino acid fragment (row d). Extending the length of the C-terminal polypeptide to 85 and 101 amino acids visibly improved the strength of the interaction, as shown in Fig. 2 (rows h and g). Conversely, smaller deletions that removed the extreme C-terminal sequences failed to maintain the two-hybrid interaction (rows i–k). However, the C-terminal 30 amino acids of Nhx1 alone failed to show significant interaction (not shown). Based on these data, we conclude that the extreme C-terminal sequences (about 30 amino acids) of Nhx1 are important but not sufficient for the two-hybrid interaction with Gyp6. It is likely that the intact three-dimensional structure of the C terminus of Nhx1, rather than a discrete sequence motif, mediates binding to Gyp6.



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  		FIG. 2.
Two-hybrid interaction between Nhx1 and Gyp6. A, equal numbers of cells from freshly grown yeast cultures were applied (from left to right; undiluted, 1:10, and 1:100 dilutions) on synthetic complete (SC) medium or on selective medium (SC plus X-{alpha}-Gal, lacking supplements), as shown. Rows a–c are negative controls, showing lack of growth on selective medium of strains transformed with plasmid pGAD-NHX1515–633 alone, pGBD-GYP6271–459 alone, and pGAD-C1 empty vector with pGBD-GYP6271–459, respectively. Row d shows growth and blue coloration of colonies transformed with pGAD-NHX1515–633 and pGBD-GYP6271–459. Rows e–k correspond to deletions of the Nhx1 tail as shown. B, a topological depiction of Nhx1 (top) and deletion constructs (bottom) tested by the two-hybrid assay. The strengths of the two-hybrid interactions are summarized on the far right.

 
Membranes derived from yeast expressing full-length Gyp6 tagged at the N terminus with His6 and C-terminally HA-tagged Nhx1 were solubilized and bound to Ni-NTA beads as described under "Materials and Methods." The eluates were separated by SDS-PAGE and probed with anti-His and anti-HA antibodies to identify Gyp6 and Nhx1, respectively. As shown in Fig. 3, Nhx1-HA was pulled down with His-Gyp6 on the Ni-NTA column, confirming a physical interaction between the two proteins.



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  		FIG. 3.
His-Gyp6 physically associates with Nhx1-HA. Membranes from yeast strains transformed with plasmids expressing His-Gyp6 and Nhx1-HA, separately or together as shown, were solubilized, bound to Ni-NTA beads, and washed thoroughly, as described under "Materials and Methods." Eluates were separated by SDS-PAGE and subjected to Western analysis using anti-His (top) and anti-HA antibodies (bottom). Nhx1-HA coeluted with His-Gyp6, as shown in the leftmost lane.

 
Gyp6 Is a Membrane-associated Protein That Colocalizes with Nhx1
Of the six Gyp family members that have been described so far in yeast (21), little is known about Gyp6 other than its substrate preferences in vitro (30). We reasoned that colocalization of Gyp6 and Nhx1 in the same membrane compartment would allow a putative functional interaction between the two proteins. Although hydropathy analysis predicts no transmembrane spans in Gyp6, we show here that it is a membrane-associated protein. Following ultracentrifugation of total cell extracts in 10 mM potassium phosphate buffer (see "Materials and Methods"), His-Gyp6 was entirely associated with the membrane pellet, similar to that observed for Nhx1-HA. To determine the nature of the membrane association, total membrane fractions were washed under a variety of conditions designed to remove peripherally associated proteins, i.e. low ionic strength (20 mM EDTA), high salt (300 mM KCl or 600 mM NaCl), or high pH (50 mM Na2CO3, pH 11). These treatments were able to extract significant amounts of Gyp6 from the pellet to the supernatant fractions (Fig. 4). In contrast, Nhx1 remained in the pellet, as expected for an integral membrane protein. The membrane association of Gyp6 was not altered in the {Delta}nhx1 mutant (not shown). Interestingly, treatment of membranes with 1% Triton X-100 at 0 °C efficiently extracted Nhx1 but not Gyp6. Because proteins associated in lipid rafts are characterized by insolubility in cold Triton X-100, it is possible that Gyp6 localizes to raft domains. Similar results have been reported for the membrane-associated Gyp1 protein (31).



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  		FIG. 4.
His-Gyp6 associates with membranes. Total membrane preparations (100 µg) from yeast expressing His-Gyp6 and Nhx1-HA were incubated on ice for 15 min under the conditions shown followed by high speed centrifugation as described under "Materials and Methods." Supernatant (S) and pellet (P) fractions were subjected to SDS-PAGE and Western analysis with anti-His antibody (top) and anti-HA antibody (bottom).

 
Gyp1 and Gyp5 have been shown to localize to the Golgi and cytosol, respectively, whereas Gyp8 was recently localized to a compartment distinct from the Golgi, resembling the PVC (21, 31). In preliminary studies using confocal microscopy, we found that GFP-tagged Gyp6 appeared as one or two intensely fluorescent spots/cell, next to the vacuole (not shown). This was reminiscent of our previous observations with Nhx1-GFP (4) and suggested that Gyp6, like Nhx1, localized to the prevacuolar compartment in yeast. Fig. 5 confirms the colocalization of CFP-Gyp6 with GFP-Nhx1 in the PVC, which was stained with FM4-64 according to the protocol of Shin et al. (24). It is noteworthy that we did not see any obvious change in localization of Gyp6 in the {Delta}nhx1 mutant or vice versa (not shown), arguing against a scaffolding function for the Nhx1 tail in mediating membrane attachment or localization of Gyp6. We also found that CFP-Gyp8 does indeed localize to the PVC, along with GFP-Gyp6 (not shown).



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  		FIG. 5.
Endosomal localization of Gyp6. Fluorescence microscopy of live yeast cells showing the colocalization of Nhx1-GFP (green, A) with CFP-Gyp6 (blue, B), with FM4-64 stain of the PVC (red, C). An overlay of the three images is shown in D.

 
Gyp6 Has Novel Trafficking and pH Phenotypes That Overlap with Nhx1
Although the GAP activity of Gyp6 is well documented in vitro, there are no phenotypes reported on the deletion or overexpression of Gyp6, and little is known about the physiological role of this protein. A purified glutathione S-transferase fusion of Gyp6 was shown to activate GTP hydrolysis of Ypt6, suggesting a role in Ypt6-mediated retrograde trafficking to the Golgi (16, 30). However, significant GAP activity was also observed with purified Ypt7, which is involved in PVC to vacuole trafficking (30). Intriguingly, deletion of NHX1/VPS44 was previously shown to disrupt trafficking out of the PVC (10). We therefore hypothesized that the role of Nhx1 in vesicular traffic may be mediated in part by its interaction with Gyp6, particularly in light of our finding that these two proteins colocalize in the PVC. Here, we show four novel phenotypes associated with Gyp6 which demonstrate its role in vesicular trafficking along the Golgi-PVC-vacuole pathway in yeast and are consistent with a functional interaction with Nhx1.

Gyp6-overexpressing Cells Show a CPY Missorting Defect—It is well known that vacuolar protein sorting (vps) mutants display an aberrant secretion of CPY and other vacuolar hydrolases into the extracellular medium (32, 33). Thus, in a colony overlay assay (Fig. 6), the nhx1/vps44 mutant shows strong immunostaining for secreted CPY, consistent with previous reports (10). Negligible amounts of CPY were secreted in wild type (Fig. 6) as well as the gyp6 null mutant (not shown). We show that CPY secretion in the nhx1 mutant is clearly ameliorated by additional deletion of Gyp6. This observation is consistent with the removal of a negative regulator of Ypt6- or Ypt7-dependent vesicle fusion so that the delayed exit out of PVC resulting from the loss of Nhx1 can be partially compensated by efficient fusion of the vesicles with the recipient compartment. Conversely, we found that moderate overexpression of Gyp6, under control of the PGK promoter in a multicopy plasmid, resulted in increased CPY secretion over wild type levels (Fig. 6). The Gyp6 overexpression phenotype is consistent with inhibition of Nhx1-regulated vesicle budding from the PVC, as well as inhibition of Ypt6- or Ypt7-regulated vesicle fusion at the Golgi or vacuole, respectively.



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  		FIG. 6.
Effect of Gyp6 on CPY secretion. Equal numbers of cells from freshly grown yeast cultures were deposited as undiluted, 1:2, 1:5, or 1:10 dilutions (top to bottom) on a nitrocellulose filter overlaid on SC plates, incubated, and washed as described under "Materials and Methods." Extracellular CPY secreted from colonies of wild type, isogenic null mutants, or the Gyp6-overexpressing strain (GYP6) was detected by immunostaining with anti-CPY antibody. A, densitometric scan of the immunoblot, shown in B.

 
Gyp6 Expression Alters Resistance to Hygromycin B—The aminoglycoside antibiotic, hygromycin B inhibits the growth of prokaryotic and eukaryotic cells by inhibiting protein synthesis (34, 35). Earlier, we reported that the nhx1 null mutant is extremely sensitive to low concentrations of hygromycin (5). We show here that ypt6 and ypt7 mutants are also more sensitive to hygromycin, suggesting that hygromycin sensitivity may be common to trafficking mutants in the Golgi to vacuole pathway (Fig. 7A). Indeed, Yoshida and Anraku (36) have shown that Class C and D vps mutants are hygromycin-sensitive. Interestingly, the gyp6 null mutant shows enhanced tolerance to hygromycin, and conversely, overexpression of Gyp6 in the wild type strain resulted in increased hygromycin sensitivity. Again, these findings are consistent with Gyp6 being a negative regulator of the Golgi-PVC-vacuole trafficking pathway. It has been suggested that Gyp proteins play a redundant role in the cell because of their promiscuity toward substrates documented in vitro. For example, Gyp8 also exhibits GAP activity toward Ypt6 in vitro (21), and its localization to the PVC suggests that it may have a functional overlap with Gyp6. However, we show that the gyp8 mutant exhibits no change in hygromycin sensitivity, supportive of the specificity of interaction between Gyp6 and its physiological partners in vivo.



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  		FIG. 7.
Functional interaction between Gyp6 and Nhx1. A, hygromycin sensitivity. Wild type (WT), isogenic deletions, or Gyp6-overexpressing yeast (GYP6) were grown for 24 h at 30 °C in APG medium, pH 4, supplemented with 25 µg/ml hygromycin B. Growth was measured as A600 nm and expressed in each case as a percent of control cultures containing no hygromycin B. The average of duplicate measurements from three independent experiments is shown. B, plasma membrane potential. The accumulation of [14C]methylammonium into wild type or isogenic deletion strains was quantitated after 40 min of incubation at 23 °C as described under "Materials and Methods." The average of triplicate measurements from one of three representative experiments is shown. C, vacuolar pH. Fluorescence intensity at 490 nm was measured in BCECF-loaded cells grown in APG medium, pH 2.7, and normalized to cell density as described under "Materials and Methods." The absolute values of vacuolar pH shown were estimated after calibration (Fig. 1). The average of 6–18 independent experiments is shown.

 
Loss of Plasma Membrane Potential in the gyp6 Null Mutant—Changes in the plasma membrane electrical potential are known to alter sensitivity to hygromycin and other toxic cations by influencing their uptake. Thus, decreased H+ pumping activity at the plasma membrane, as seen in pma1 mutations or in the {Delta}ptk2 null strain, depolarizes the membrane potential and confers tolerance to toxic cations, including Na+, Li+, Mn2+, methylammonium, hygromycin B, and norspermidine (37, 38). In fact, we found that addition of 400 mM KCl effectively protected yeast from hygromycin toxicity, consistent with a depolarizing effect of elevated external K+ on the membrane potential (not shown and Ref. 39). Conversely, null mutants in the Trk H+,K+ cotransport system have been shown to hyperpolarize the plasma membrane and increase sensitivity to toxic cationic compounds (40). We quantitated uptake of [14C]methylammonium in an isogenic set of wild type and mutant yeast as an indicator of membrane potential. As described earlier, uptake of this cationic compound was increased in {Delta}trk1 and decreased in {Delta}ptk2, consistent with hyperpolarization and depolarization of the membrane potential, respectively (37, 40). In three independent experiments, we observed a reproducible increase in uptake of [14C]methylammonium in {Delta}nhx1 and Gyp6-overexpressing cells, and a corresponding decrease in {Delta}gyp6 cells (Fig. 7B). Uptake was only slightly increased in ypt6 and ypt7 null strains (not shown). These changes in membrane potential are likely to contribute toward the observed sensitivities to hygromycin B in these mutants. However, it appears that Golgi-to-vacuole trafficking must also play an important role in determining hygromycin sensitivity. This would explain hygromycin sensitivity of the ypt mutants (Fig. 7A) and our observation that hygromycin sensitivity in the nhx1 mutant is much greater than in the trk1 mutant (not shown) despite more modest changes in membrane potential. We suggest that vacuolar sequestration of hygromycin is compromised in Golgi-to-vacuole trafficking mutants and that this contributes to the sensitive phenotype.

Alkalinization of Vacuolar pH in the gyp6 Null Mutant—The acidic nature of the yeast vacuole is essential for its function in storage, degradation, and detoxification of metabolites (41). Earlier, we had proposed that the Na+/H+ exchange activity of Nhx1 serves as a "leak" pathway for protons out of the endosome, limiting intraorganellar acidification by the vacuolar H+-ATPase in prevacuolar compartments (4). Indeed, Bowers et al. (10) showed that the nhx1 null strain exhibited hyperaccumulation of the dye FM4-64 in the PVC and an inappropriate proteolysis of the Vps10 protein, consistent with increased acidity of the endosomal lumen. In this work, we take advantage of the vacuolar sequestration of the pH-sensitive fluorescent dye, BCECF (Fig. 1 and "Materials and Methods"), to compare luminal pH in a set of isogenic yeast strains. As predicted, in the nhx1 null mutant, vacuolar pH was significantly more acidic than wild type in BCECF-loaded cells subjected to acid stress (APG medium, pH 2.7). The changes in vacuolar pH correlated with a growth sensitivity of nhx1 in acidic medium (27 ± 3% growth, relative to wild type at pH 2.7), suggesting that excessive acidification of the vacuole was not compatible with growth. Strikingly, vacuolar pH in the gyp6 mutant was substantially more alkaline (Fig. 7C) and correlated with improved growth at pH 2.7 (109 ± 4% growth relative to wild type). The gyp8 mutant did not show consistent differences in either vacuolar pH (Fig. 7C) or growth sensitivity (not shown) relative to wild type, again supporting the hypothesis that Gyp6 has specific cellular functions, different from those of Gyp8. We show that in acid-stressed cells, vacuolar pH in ypt6 and ypt7 null mutants, and in Gyp6-overexpressing cells, was significantly more acidic than wild type. It should be noted that vacuolar morphology was aberrant in the latter three strains (42, 43, and Fig. 1, inset), with a variable fragmentation/vesiculation that was most severe in the ypt7 mutant. However, this did not preclude compartmental accumulation of the fluorescent dye.

In summary, increased hygromycin tolerance, increased vacuolar pH, and decreased plasma membrane potential in the gyp6 mutant are consistent with increased Nhx1 activity. Taken together with the increased hygromycin sensitivity and increased CPY secretion observed in the Gyp6-overexpressing strain, our findings suggest that Gyp6 may be a negative regulator of Nhx1 activity. Increased availability of this negative regulator in the ypt6 and ypt7 deletion strains may account for the nhx1-like phenotypes of the latter because Gyp6 may interact with these two proteins in vivo as an activator of GTP hydrolysis (15).

Ypt6 and Nhx1 Function Along the Same Trafficking Pathway
We show here that Nhx1 and Ypt6 share cellular phenotypes of increased secretion of CPY and increased sensitivity to hygromycin B, indicating that they function along the same trafficking pathway. To explore further the relation between these two proteins in the PVC to Golgi pathway, we examined the effects of combinations of mutations in YPT6 and NHX1. We found that CPY secretion defect in the nhx1 mutant is more severe than in ypt6 (not shown). Fig. 8A shows that the CPY secretion defect of ypt6 is improved significantly by the additional deletion of nhx1, but not by overexpression of Nhx1. Next, we examined the effect of NHX1 disruption on the temperature-sensitive growth phenotype of ypt6. Fig. 8B shows that the nhx1 mutant shows no temperature-sensitive growth phenotype, whereas the ypt6 mutant shows reduced growth at 37 °C, as reported previously (45). Disruption of NHX1 significantly improves growth of ypt6 at 37 °C in synthetic medium. Taken together, our data suggest that a decrease in the traffic out of the PVC, resulting from disruption or inhibition of Nhx1, relieves a downstream vesicle fusion defect resulting from the ypt6 mutation.



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  		FIG. 8.
Functional interaction between Nhx1 and Ypt6. A, secretion of CPY, from colonies overlaid on nitrocellulose filters, was detected as described in the legend to Fig. 6. Freshly grown cultures of wild type (WT), mutants, or Nhx1-overexpressing cells were applied from left to right as follows: undiluted, 1:2, 1:5, and 1:10 dilutions. B, equal numbers of cells from wild type or isogenic mutants were applied on to synthetic complete medium and incubated for 24 h at 30 or 37 °C, as shown. From left to right, undiluted, 1:10, and 1:100 dilutions of cultures.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nhx1 is the founding member of an evolutionarily conserved family of intracellular Na+/H+ exchangers, distributed in microorganisms, plants, and animals (7). Although related to the plasma membrane NHE, Nhx1 homologs localize to endosomal/vacuolar compartments where they are important in salt and osmotolerance, compartmental pH regulation, and vesicular trafficking. In yeast, the nhx1 null mutant shows an accumulation of vacuolar and Golgi markers in an aberrant class E compartment and is sensitive to salt and osmotic stress (10, 46). These phenotypes implicate a role for intraluminal ion concentration and pH in vesicle sorting and trafficking, although the factor(s) that link these two vital biological functions remain to be elucidated. In the present study, we have identified one such factor by yeast two-hybrid screening and provided evidence supporting a functional interaction that can synchronize events along the trafficking pathway.

We show that the C-terminal domain of Nhx1 physically interacts with a C-terminal region of Gyp6, outside its catalytic GAP domain. The phenotypes of the gyp6 null mutant and Gyp6 overexpressing strain suggest that Gyp6 may be a negative regulator of Nhx1. Thus, we show that in a gyp6 null mutant hygromycin tolerance is enhanced, vacuolar pH is elevated, and membrane potential is decreased, consistent with increased Na+/H+ exchange at the endosomal membrane. Conversely, overexpression of Gyp6 results in increased secretion of CPY, increased sensitivity to hygromycin B, and acidification of the vacuole, similar to loss-of-function phenotypes of Nhx1. A null mutant of Gyp8, another Ypt6-GAP that colocalizes with Gyp6, does not show many of these phenotypes, suggesting that effects on Nhx1 activity are specific to Gyp6. Gyp6 has also been shown to interact with Ypt6, stimulating the low intrinsic GTPase activity of the latter (0.0002 min-1) by a factor of 5 x 106 (30). Because the GTP-bound form of Ypt6 is required for activation of vesicle docking and fusion, the GAP activity of Gyp6 must serve as a negative regulator of the Ypt6-mediated pathway of retrograde traffic from the endosome to the Golgi/TGN. Taken together, these observations suggest an inhibition of retrograde traffic by the Gyp6-bound state of both Nhx1 and Ypt6. Further studies will be needed to separate the individual effects of Gyp6 on its binding partners in the context of vesicular trafficking.

In this study we demonstrate the membrane association of Gyp6 and its localization to the prevacuolar compartment. This places Gyp6 in position to provide GAP activity for Ypt6, consistent with its preference for Ypt6 in vitro (30). In previous studies, GEF proteins have been shown to colocalize with their cognate Ypt substrates at the site of vesicle fusion, i.e. acceptor compartment. For example, the GEF protein Sec2 colocalizes with the Ypt protein Sec4 at the exocyst (49), and the Ric1/Rgp1 complex colocalizes with Ypt6 at the Golgi (43). Because Ypt6 has been implicated in retrograde traffic from the endosome to Golgi, the placement of Gyp6 at the endosome/donor compartment is consistent with its role as a Ypt6 GAP. However, our studies do not exclude the possibility that Gyp6 also exerts GAP activity on Ypt7 because the endosome also serves as the donor compartment for Ypt7-regulated endosome-tovacuole anterograde trafficking. Because both Ypt6 and Ypt7 share phenotypes of CPY secretion and hygromycin B sensitivity, the observed effects of Gyp6 could reflect an interaction with either or both Ypt6- and Ypt7-mediated pathways. It will be necessary to examine Ypt6- and Ypt7-specific phenotypes to clarify the role of Gyp6. In any case, our studies indicate that Gyp6 serves as a negative regulator of Ypt-regulated vesicle fusion events. Thus, we show that overexpression of Gyp6 mimics the effects of ypt mutations, and conversely the gyp6 null mutant relieves trafficking defects, albeit slightly, in the nhx1 null mutant. Interestingly, the lack of phenotypes relating to hygromycin B tolerance or vacuolar pH in the gyp8 mutant is evidence that it may have a role different from that of Gyp6 in vivo.

The significance of our findings is illustrated in the model depicted in Fig. 9. At the prevacuolar compartment, cargo destined for anterograde movement to the vacuole, must be sorted from retrograde traffic to the Golgi/TGN. We propose that vesicles with increasing acidity are targeted to the vacuole, and conversely, vesicles that are less acidic are targeted for retrograde traffic, consistent with the experimental observations of Stoorvogel and colleagues (2, 3). In this scenario, Nhx1 inhibition by Gyp6 would generate acidic vesicles destined for vacuolar delivery while concomitantly inhibiting retrograde delivery. If Ypt6 and Nhx1 compete for binding of Gyp6, then recruitment of Gyp6 by Ypt6-GTP at the prevacuolar membrane would release Nhx1 from inhibition, alkalinizing the vesicle lumen and promoting retrograde traffic. This would have the effect of returning Ypt6-GDP to the Golgi, where it can be reactivated by the Ric1/Rgp1 (GEF) complex prior to mediating proper vesicle docking and fusion. The model proposes a coordination of events at the donor (PVC) and acceptor (TGN/Golgi) compartments by a common inhibitor, Gyp6. Thus, factors or cellular events that result in decrease in anterograde traffic to the PVC would concomitantly cause a decrease in retrograde traffic to the TGN/Golgi. This is consistent with our data showing that a defect in vesicle fusion at the Golgi, resulting from loss of Ypt6, can be partially compensated by a reduction in traffic out of the PVC, caused by disruption of Nhx1. There is new evidence for coordinated regulation of events in trafficking pathways. For example, Ypt/Rab proteins have been shown to act in a cascade such that activation of one Ypt protein results in stimulation of another Ypt protein downstream in the trafficking pathway. Thus, the activated form of Ypt1 influences the activity of Ypt32 by recruiting the Ypt32 GEF factor, directing traffic to and through the Golgi complex (47). Similarly, activated Ypt32 recruits the GEF for the Ypt protein Sec4 along the Golgi-to-plasma membrane pathway (48). Our study suggests that coordinated inhibition of upstream and downstream components in a trafficking pathway may also serve to synchronize events.



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  		FIG. 9.
Model for regulation of endosomal traffic by Gyp6. At the PVC/endosome, Gyp6 inhibits Nhx1 activity and limits retrograde traffic to the TGN/Golgi. Under these conditions, vesicles are more acidic, and anterograde traffic predominates (anterograde). Upon delivery to the PVC, Ypt6-GTP competes with Nhx1 for Gyp6 binding. The Ypt6-Gyp6 interaction serves to relieve Nhx1 inhibition, alkalinize the vesicle, promote retrograde traffic, and terminate the Ypt6 signal by GTP hydrolysis (retrograde). Ypt6-GDP returns to the TGN/Golgi where it is reactivated by the Ric1/Rgp1 GEF and directs vesicle fusion.

 
Several features of this model are amenable to testing and further investigation. Although it is not known whether Ypt6 traffics to the PVC, the location of Gyp6, its cognate GAP protein, in this compartment implies a continuous cycling of Ypt6 between the Golgi and prevacuolar compartments. Small N- or C-terminal deletions in Gyp6 have been shown to disrupt interaction with Ypt6 completely (30), suggesting that binding of Nhx1 and Ypt6 to Gyp6 may be mutually exclusive, as we predict. A prediction of the competitive binding model is that deletion of Ypt6 would increase Gyp6-mediated inhibition of Nhx1, consistent with the observed vacuolar pH acidification in the {Delta}ypt6 strain. At the present time, we do not know whether Gyp6 binding to Nhx1 in vivo is itself affected by vesicle acidification, as has been shown for Arf6 and ARNO binding to unknown receptor(s) on endosomal vesicles from the proximal tubule (14). This would not be unexpected, given that the C-terminal domain of NHE is known to modulate the pH-dependent activation of the exchanger. It is possible that Nhx1 itself serves as a pH sensor, by altering the conformation of the C-terminal domain in response to luminal pH.

We provide new evidence of Nhx1 function in regulating vacuolar pH and plasma membrane potential. The existence of leak pathways for protons has been proposed to be the principal mechanism of limiting pH gradient in compartments en route to the vacuole/lysosome. Such a mechanism would play an important role in preventing excessive acidification by the vacuolar H+-ATPase, which is widely distributed in Golgi, TGN, and endosomal membranes. The inhibition of growth and increased acidification of vacuolar/endosomal lumen in the nhx1 mutant in response to low pH stress illustrate the importance of vacuolar pH homeostasis in supporting growth. Our findings are complementary to the work of Plant et al. (25), who have demonstrated increased alkalinity in the vacuolar ATPase mutants which correlates with an inability to grow in high pH media. We also demonstrate a complex interrelation between H+ circuits at the vacuole and plasma membrane. The transport of H+ into the cytosol by Nhx1 would be expected to decrease the H+ gradient generated by the plasma membrane H+-ATPase Pma1 and limit development of the plasma membrane potential. This would explain the hyperpolarization of the plasma membrane observed in the nhx1 mutant, demonstrated by increased uptake of the cationic compound [14C]methylammonium. Our data also suggest that hygromycin sensitivity in the nhx1 mutant is at least partly the result of increased uptake of hygromycin.

In conclusion, this study suggests a novel, coordinated regulation of the retrograde vesicle trafficking pathway from the PVC/endosome to Golgi. Two effectors of this pathway, Ypt6 and Nhx1, appear to be regulated by Gyp6 through protein-protein interaction, indicating a specific role for Gyp6 in vesicle trafficking.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK54214 (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Predoctoral fellow of the American Heart Association, Mid-Atlantic Affiliate. Back

§ To whom correspondence should be addressed: Dept. of Physiology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-4732; Fax: 410-955-0461; E-mail: rrao{at}jhmi.edu.

1 The abbreviations used are: TGN, trans-Golgi network; BCECF, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; CPY, carboxypeptidase Y; GAP, GTPase-activating protein; GEF, GTP/GDP exchange factor; GFP, green fluorescent protein; HA, hemagglutinin; MES, 4-morpholineethanesulfonic acid; NHE, Na+/H+ exchanger; Ni-NTA, nickel-nitrilotriacetic acid; PVC, prevacuolar compartment. Back


   ACKNOWLEDGMENTS
 
We thank Van-Khue Ton for assistance with confocal microscopy, Beverly Wendland for assistance with the CPY assay, Dieter Gallwitz for the CFP-GYP8 construct, Susan Michaelis for strain BY4741, and Andy Hoyt for the yeast genomic library and two-hybrid vectors. We also thank Susan Michaelis and Mark Donowitz for a critical reading of this manuscript.



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