Article |
Address correspondence to Peter J. Novick, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. Tel.: (203) 785-5871. Fax: (203) 785-7446. E-mail: peter.novick{at}yale.edu
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Abstract |
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Key Words: membrane traffic; Ypt31/32; exchange factor; Rab; yeast
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Introduction |
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Transport of vesicles from the Golgi apparatus to the plasma membrane has been studied in many cell types. In yeast, Golgi apparatusderived vesicles are transported to sites of polarized growth, including tips of small buds and motherdaughter necks. This process relies on the actin cytoskeleton and Myo2p, a class V myosin (Novick and Botstein, 1985; Govindan et al., 1995; Pruyne et al., 1998; Schott et al., 1999; Karpova et al., 2000). Cells treated with the actin polymerization inhibitor latrunculin A or harboring mutations that affect either actin function (act1-1) or Myo2p function (myo2-66) accumulate secretory vesicles randomly throughout the cell. The polarized delivery of post-Golgi vesicles also depends on the functions of Sec4p and Sec2p. Sec4p is the Rab protein that regulates this stage of the secretory pathway and Sec2p is the GEF that activates Sec4p (Salminen and Novick, 1987; Walch-Solimena et al., 1997). Sec2p and Sec4p are found in association with secretory vesicles and therefore localize to sites of polarized secretion. Temperature-sensitive (ts) mutants defective in most of the genes required for Golgi to plasma membrane transport accumulate a cluster of secretory vesicles adjacent to normal exocytic sites at the restrictive temperature, indicating that vesicle delivery was not blocked. However, ts mutations in sec2 cause an accumulation of vesicles randomly distributed throughout the cell, similar to that seen in act1-1, myo2-66, and latrunculin Atreated cells. This suggests that activation of Sec4p by Sec2p is necessary for the vectorial transport or retention of vesicles at sites of secretion. There are several examples in which Rab proteins interact with components of the actin or microtubule cytoskeleton to regulate membrane traffic (Echard et al., 1998; Hume et al., 2001; Lapierre et al., 2001). However, the only known effector for Sec4p is Sec15p, a component of the exocyst complex, which is necessary for tethering secretory vesicles to exocytic sites (Guo et al., 1999). This implies that there may yet be an unidentified effector of Sec4p that mediates the polarized transport of vesicles.
The mechanism by which Sec2p binds to secretory vesicles is not understood. Sec2p does not contain transmembrane domains or lipid modifications that would anchor it to the vesicle membrane and, moreover, the association of Sec2p with secretory vesicles is partially independent of its interaction with Sec4p (Elkind et al., 2000). Sec2p contains a stretch of 58 amino acids (aa 450508) that is necessary but not sufficient for its association with secretory vesicles. Mutations or truncations of this domain give rise to ts alleles that disrupt the ability of Sec2p to bind secretory vesicles, but do not affect its exchange activity. Based on this evidence, we hypothesize that a Sec2p receptor must exist on secretory vesicles.
Here we report the results of a high copy suppressor screen for either effectors of Sec4p involved in polarized transport or membrane receptors for Sec2p. We screened for genes whose overexpression can suppress the growth defect of sec2-78 at 37°C. The sec2-78 allele has a point mutation changing amino acid 483 from a cysteine to a tyrosine within the 58amino acid stretch that is critical for proper localization of the protein. Two of the suppressors identified in this screen, YPT31 and YPT32, encode functionally redundant Rab proteins implicated in intra-Golgi transport and budding of secretory vesicles from the Golgi apparatus (Benli et al., 1996; Jedd et al., 1997). We demonstrate that overexpression of Ypt32p restores the localization of two mutant Sec2 proteins to exocytic sites. We also show through biochemical studies that Ypt32p binds to Sec2p preferentially in its GTP-bound state. Based on these results, we propose that Ypt32p and Sec4p together through their interaction with Sec2p regulate delivery of post-Golgi vesicles to the plasma membrane.
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Results |
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To confirm that overexpression of Ypt31p and Ypt32p suppress the sec2-78 mutation, cells were transformed with a plasmid containing YPT31 and hemagglutinin (HA) epitopetagged YPT32 under the control of the GAL1 promoter. Overproduction of Ypt31p and Ypt32p was confirmed by Western blotting with antibodies against Ypt32p (Fig. 1 A). We also transformed the cells with HAYPT1 and HASEC4 under the control of the GAL1 promoter. Fig. 1 B shows a Western blot using an anti-HA antibody (12CA5). The constructs were expressed at levels similar to HAYPT32. We then grew the cells on YP plates containing either glucose (YPD) or a mixture of galactose and raffinose (YPGR), at different temperatures (25°C, 34°C, and 37°C). As shown in Fig. 1 C, wild-type cells grow well at all temperatures, wheras sec2-78 cells were unable to grow at 37°C on YPD or YPGR. However, when Ypt31p, Ypt32p, or Sec4p were overexpressed, growth at the restrictive temperature (37°C) was restored. Ypt1p was unable to suppress the sec2-78 growth defect. We also tested whether overexpression of these proteins was able to suppress another sec2 allele, sec2-59. This mutant lacks most of the COOH terminus and is unable to grow at 34°C or 37°C. Overexpression of Ypt31p and Ypt32p was able to restore growth of sec2-59 at 34°C but not at 37°C (Fig. 1 C; unpublished data). However, under these conditions, Ypt1p and Sec4p were unable to suppress the growth defect of sec2-59 at either temperature.
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Overexpression of Ypt32p restores the localization of Sec2-78pGFP
In wild-type yeast, Sec2p associates with secretory vesicles. As a result, Sec2pgreen fluorescent protein (GFP) localizes to exocytic sites, bud tips, and motherdaughter necks. Mutations or truncations in the COOH terminus, such as Sec2-78p and Sec2-59p, cause the protein to dissociate from secretory vesicles, resulting in a diffuse distribution throughout the cytoplasm (Walch-Solimena et al., 1997; Elkind et al., 2000).
To study the effects of Ypt31p and Ypt32p overexepression on the localization of Sec2-78pGFP, we used strains NY2429 (sec2-78GFP, GAL-YPT32) and 2430 (sec2-78GFP, GAL-YPT31). All strains in this experiment were grown at 25°C on YPgalactose (YPGal) to induce expression of the GAL1 constructs. This was confirmed by Western blotting using anti-Ypt31/32p antibodies (unpublished data). Wild-type Sec2pGFP showed characteristic localization to bud tips and necks, whereas Sec2-78p and Sec259pGFP cells exhibited diffuse cytoplasmic staining as previously described (Elkind et al., 2000; Fig. 2 A). When Ypt31p and Ypt32p were overproduced, the localization of Sec2-78pGFP was substantially restored to bud tips and necks. Localization of Sec2-59pGFP was also restored to sites of secretion upon overproduction of Ypt32p (Fig. 2 A). When these experiments were performed at 37°C, some loss of polarized localization was observed even in wild-type cells and no restoration of Sec2-78pGFP was seen by Ypt32p overexpression (unpublished data). Previous studies showed that only small amounts of full-length Sec2p are sufficient to suppress the growth defect of various sec2 alleles (Nair et al., 1990). It is possible then that at 37°C, only small amounts of protein are correctly localized to bud tips and necks and can't be detected by fluorescence. However, these small amounts of protein would be enough to support growth.
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In contrast, overexpression of Sec4p from the GAL promoter did not cause any relocalization of Sec2-78pGFP (Fig. 2 C), even though it was able to suppress the growth defect of sec2-78 at nonpermissive temperatures (Fig. 1 C). Overexpression of Ypt1p had no effect on the localization of Sec2-78p and Sec2-59pGFP, in agreement with its inability to suppress the growth defects of these mutants at the nonpermissive temperatures (unpublished data; Fig. 1 C).
Overexpression of Ypt32 restores the localization of Sec4p in sec2-78 mutant cells
In wild-type cells Sec4p, like Sec2p, localized to sites of polarized secretion. In sec2-78 cells, Sec4p was partially localized to bud tips and necks at the permissive temperature (25°C), but this localization was lost upon shifting to 37°C (Walch-Solimena et al., 1997; Fig. 3). Because overexpression of Ypt32p restores localization of Sec2-78pGFP, we investigated whether it could also restore Sec4p localization in sec2-78 cells at 37°C. For these experiments we used a 2µ plasmid with the constitutive promoter GPD to overexpress Ypt32p. We were unable to use the GAL-YPT32 construct because our immunofluorescence protocol does not work well on cells that have been grown on galactose. Strains NY10 (WT), NY2179 (sec2-78), and NY2431 (sec2-78, 2µ GPD-YPT32) were used to observe Sec4p localization (Fig. 3 A).
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Two-hybrid assay
The yeast two-hybrid system was used to explore possible interactions between Sec2p and Ypt32p (Chien et al., 1991). We made various point mutations in YPT32 based on homology with other small G proteins like Ras and Sec4p (Walworth et al., 1989). These alleles were fused with the GAL4 DNA binding domain in the pAS-CYH2 plasmid. SEC2 was fused to the GAL4 DNA activation domain in pACTII. The SEC2 construct was then screened against the YPT32 constructs. Expression of the fusion proteins was confirmed by Western blotting with anti-HA antibody (12CA5), which recognizes the HA epitope immediately adjacent to the GAL4 domain. All proteins were expressed at similar levels (unpublished data).
Ypt32S27N contains a substitution of serine for asparagine that is analogous to the dominant blocking allele RasN17. This mutant binds GDP preferentially but probably adopts a conformation intermediate between GDP- and GTP-bound states (Farnsworth et al., 1991). The Ypt32E49Q mutant exhibits cold sensitivity, and this position has been reported to be part of the effector domain (Matsuda et al., 2000). The Ypt32Q72L mutation lowers the intrinsic rate of GTP hydrolysis as demonstrated in other Rab proteins (Der et al., 1986). Ypt32N126I contains an asparagine to isoleucine mutation in the highly conserved NKXD box. This results in a protein that is unable to bind nucleotides (Walter et al., 1986). Ypt32Cys contains a deletion of COOH-terminal cysteines that are necessary for geranylgeranylation and membrane attachment of the protein.
The results of the two-hybrid assay are shown in Table I. Sec2p was found to interact with wild-type, Ypt32E49Q, Ypt32Q72L, and Ypt32Cys, but not with Ypt32S27N and Ypt32N126I. The interaction between Sec2p and the Ypt32p constructs was weaker than that observed for Ypt32p with GDI and Sec2p with Sec4S34N (preferentially binds GDP; unpublished data). This could indicate that the interaction between Ypt32p and Sec2p is a weak or transient interaction.
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In the binding experiment, GSTSec2p attached to glutathione-Sepharose beads was incubated with purified Ypt32p. Binding of GSTSec2p to three different conformations of Ypt32p, the nucleotide-free, GDP-bound, and GTP--Sbound forms, was examined. GST alone bound to the beads was used as a control for nonspecific binding. As shown in Fig. 4, all three forms of Ypt32p were able to bind to Sec2p. The apparent affinity of Sec2p for the GTP-
-Sloaded form of Ypt32p was somewhat higher than its affinity for the other nucleotide states examined in the assay. However, under these experimental conditions, only
4050% of Ypt32p was being consistently loaded with the appropriate nucleotide. It is therefore possible that the difference between GTP-
-S bound and other forms is larger than it appears in the data shown. As a control, we performed an identical binding experiment with Sec4p. In agreement with our previous results (Walch-Solimena et al., 1997), Sec2p interacted preferentially with the nucleotide-free conformation of Sec4p. Weak binding to Sec4p-GDP was also detected, but no binding of activated Sec4p-GTP-
-S was seen. The efficiency of the Ypt32Sec2p binding appeared to be slightly lower than the efficiency of the Sec4pSec2p interaction. Under the same experimental conditions, four times more Sec4p bound to Sec2p than Ypt32p. This may be due to the transient nature of this interaction. Similar, but very small, amounts of endogenous Ypt32p and Sec4p copurified with GSTSec2p from yeast (unpublished data). Therefore, it is possible that when GSTSec2p is immobilized on beads, its Ypt32p binding site might be less accessible than its Sec4p binding site.
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Sec2p does not have exchange activity on Ypt32p
To assess whether Sec2p functions as a GEF for Ypt32p, an in vitro activity assay was performed (Fig. 5). Purified Ypt32p was preloaded with [3H]GDP and the time course of nucleotide release was measured. GSTSec2p, even at the concentration of 0.25 µM, did not stimulate the nucleotide release from 0.2 µM Ypt32p. On the other hand, as shown previously, substoichiometric amounts of Sec2p were sufficient to stimulate [3H]GDP release from Sec4p (Walch-Solimena et al., 1997). In our hands, with 0.2 µM Sec4p and 10 nM GSTSec2p, threefold stimulation of the GDP release rate was observed (unpublished data). A tenfold higher amount of GSTSec2p resulted in almost a 30-fold stimulation of nucleotide release from Sec4p.
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To further define the Ypt32p and Sec4p binding sites on Sec2p, expression vectors for several truncated Sec2p alleles have been constructed and the corresponding proteins were expressed as GST fusions in yeast. As shown in a schematic diagram (Fig. 7 A), the constructs included the truncated proteins encoded by the ts allele sec2-59 (aa 1374) and the point mutant sec2-78 (Sec2C483Y) as well as additional truncations Sec2(aa 1160), Sec2(aa 161759), and allele sec2-70 (aa 1508). All of the fusion proteins were purified from yeast lysates. Purification yielded bands of the appropriate molecular weights (Fig. 7 B). These constructs were immobilized on glutathione-Sepharose beads and tested for their ability to bind Sec4p and Ypt32p (Fig. 8 A). Binding of Ypt32p to all fusion proteins except GSTSec2(aa 1160)p was observed. Based on the differential binding of Ypt32p to GSTSec2-59(aa 1374)p and GSTSec2(aa 1160)p, we believe that the Ypt32p binding site on Sec2p is located within the NH2-terminal half of the Sec2p molecule, between amino acids 160 and 374. On the other hand, all of the Sec4p binding determinants appear to be located within the NH2-terminal coiled coil domain (aa 1160) of Sec2p. The domain composed of the first 160 amino acids of Sec2p is also fully sufficient for its exchange activity (Fig. 8 B). No other part of the Sec2p molecule appears to contribute to the ability of Sec2p to catalyze the nucleotide exchange on Sec4p in vitro.
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Discussion |
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Another example of coordinated GTPase function involving Ypt31/32p was recently proposed. YPT31/32 were shown to genetically interact with another family of guanine nucleotide exchangers, the Sec7 domaincontaining Arf nucleotide exchangers (Jones et al., 1999). However, at present it is not clear how these genetic interactions translate into a biochemical mechanism. Perhaps, as in the case of Sec2p, Ypt31p and Ypt32p participate in recruitment of these exchangers to the proper sites of action. Genetic interactions of ypt31 with sec4 as well as with arf1 and ypt1 alleles were also reported (Yoo et al., 1999).
The Rab protein cascade mechanism we report is strikingly similar to the mechanism by which the functions of Bud1/Rsr1p and Cdc42p, two GTPases that regulate polarity establishment and bud formation in yeast, are coordinated. They are functionally linked through the action of Cdc24p, which serves as a nucleotide exchange factor for its downstream GTPase Cdc42p. Analogous to the Ypt32pSec2p interaction, BUD1/RSR1 was isolated as a multicopy suppressor of a cdc24-4 mutation (Bender and Pringle, 1989), and Bud1/Rsr1p was subsequently shown to bind directly to Cdc24p in its GTP-bound form (Park et al., 1997). Sequential action of two GTPases was also proposed to play a role in vacuole docking and fusion (Eitzen et al., 2000).
We were able to localize binding sites on Sec2p for Ypt32p and Sec4p. Despite the fact that these two GTPases are strongly related by sequence, they bind to different domains of the Sec2 protein. Sec4p binds to the exchange domain (aa 1161), whereas the Ypt32p binding site is located further downstream (aa 161374). This is consistent with the fact that only Sec4p is a substrate for nucleotide exchange. It is important to note that truncation of Sec2p at position 374, as in sec2-59, or mutation of position 483 from cysteine to tyrosine, as in sec2-78, causes mislocalization of Sec2p. Thus, localization can be lost despite the fact that the Ypt32p binding site is intact. Nonetheless, overproduction of Ypt32p restores the localization of Sec2-59GFP or Sec2-78GFP proteins. Therefore, it appears likely that there is yet another, unidentified component regulating Sec2p localization. We propose that this component, whose identity is presently unknown, requires the region of Sec2p downstream from position 374. When this interaction is lost, localization of Sec2p can be restored by a compensatory increase in the level of Ypt32p. This situation is somewhat analogous to that of the Rab5 effector EEA1 (Simonsen et al., 1998). In that case, both Rab5 and PI3P are important for recruiting the protein to endosomes. Overproduction of one ligand can compensate for the loss of the other.
In conclusion, we have uncovered new details regarding the regulation of Sec2p function. We identified Ypt32p as a new factor involved in recruiting Sec2p to sites of polarized growth. The current data are consistent with the existence of a Rab protein cascade regulating yeast exocytosis. In this cascade, the first Rab (Ypt32p) recruits the exchange factor for its downstream Rab (Sec4p). Our data also suggest the existence of an additional factor regulating Sec2p function.
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Materials and methods |
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Plasmid construction and strains
Construction of GAL-YPT1, GAL-YPT32, and GAL-SEC4 (pNB829833) was described earlier (Grote and Novick, 1999). To create pNB1162, pNB1176, and pNB1177, YPT32, YPT1, and SEC4 (BamHI/HindIII fragments from pNB831, 829, 833) were cloned into pNB530. To create GAL-YPT31 (pNB1178), the gene was PCR amplified from genomic DNA. The product was cloned into pNB530 by digesting with BamHI/HindIII. Plasmids pNB829833 were digested with ClaI to direct integration into the leu2-3,112 gene of NY2179 (Mat leu2-3,112 ura3-52 sec2-78 GAL+) to create strains NY2440, 2427, and 2441. Plasmid pNB1178 was digested with NcoI to direct integration into the ura3-52 gene of NY26 (Mat
, ura3-52, sec2-59), NY2179, and NY2152 (Mata sec2-
1::HIS3 leu2-3,112::[LEU2 sec2-78GFP] his3-
200 ura3-52 Gal+) to create strains NY2444, 2445, and 2430. Plasmids pNB1162, 1176, and 1177, digested with NcoI, were integrated into NY26, creating strains NY2426, 2442, and 2443. pNB1162, digested with NcoI, was integrated into NY2147 (Mata sec2-
1::HIS3 leu2-3,112::[LEU2 sec2-59GFP] his3-
200 ura3-52 Gal+) and NY2152 (Mata sec2-
1::HIS3 leu2-3,112::[LEU2 sec2-78GFP] his3-
200 ura3-52 Gal+) to create strains NY2428 and 2429, whereas pNB1177, linearized with NcoI, was transformed into NY2152 to create NY2446 (Table II).
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To create the GSTSEC2 construct, DNA encoding Sec2p was PCR amplified using pNB978 as a template. The product (SEC2; 2.3 kb) was digested with BamHI and XhoI and ligated to pGEX4T-1 predigested with the same enzymes, resulting in pNB1152. PNB1152 was then used as a template for another round of PCR amplification with primers 5'-ATATCTGCAGATGTCCCCTATACTAGGTTATTGG-3' and 5'-ATATCTCGAGAAGCTTATTGCTGTTCCTGGGC-3'. The product (GSTSEC2; 3.0 kb) was digested with PstI and HindIII and ligated to pNB529 (integrating yeast vector with GAL1 promoter and ADH terminator) resulting in pNB1153. pNB1153 was linearized with ClaI and introduced into the protease-deficient pep4::HIS3 yeast strain NY603, resulting in NY2432. To induce protein expression, cells were grown in YPGal (2% galactose).
DNA regions coding for GSTSec2-59 (aa 1374), GSTSec2-70 (aa 1508), and GSTSec2(aa 1160) were amplified from pNB1152 as well. The products (GSTsec2-59, 1.8 kb; GSTsec2-70, 2.2 kb; GSTsec2(aa 1160), 1.15 kb) were digested with PstI and HindIII and ligated to pNB529, resulting in pNB1155, pNB1156, and pNB1157.
To prepare GSTsec2(aa 161759), part of the SEC2 gene was first amplified using pNB978 as a template. The product was digested with BamHI and XhoI and ligated to pGEX4T-1, resulting in pNB1158. GSTsec2(aa 161759) was then amplified from pNB1158 using the same primers described above, resulting in pNB1159. The resulting plasmids were linearized and transformed into the protease-deficient pep4::HIS3 yeast strain NY603, resulting in NY24332437 (Table II).
GFP and immunofluorescence
Cells containing Sec2 proteins tagged with GFP, with and without overexpression of Ypt31p, Ypt32p, and Sec4p were grown overnight to midlog phase (A600 = 0.51.0) in YPGal at 25°C. Cells were fixed in ice cold methanol and acetone, washed three times with phosphate buffer (PBS), sonicated for 5 s, and observed under the microscope. Sec4p was visualized in strains NY10, 2179, and 2431 by indirect immunofluorescence. Strains were grown overnight in YPD at 25°C to a final cell density (A600) between 0.5 and 1.0 U. The cells were then either immediately fixed or shifted to the restrictive temperature (37°C) for 1 h. Preparation of samples was performed essentially as described (Walch-Solimena et al., 1997). GFP and Sec4p immunolabeling were visualized with a ZEISS Axiophot 2 microscope using a 100x objective. Images were acquired with a Photometrics Quantix CCD camera.
Two-hybrid assay
S. cerevisiae strain Y190 (Mata, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, gal4-542, gal80-538, URA3::GAL-LacZ, LYS2::GAL-HIS3, cyhr) was simultaneously transformed with DNA activation and DNA binding domain constructs. Transformants were monitored for expression of ß-galactosidase activity using an X-Gal filter lift assay. For this, yeast colonies were transferred to filter paper (no. 1; Whatman Inc.), permeabilized by submerging twice in liquid nitrogen for 10 s, and then laid onto a second filter presoaked in Z buffer (100 mM sodium phosphate, 10 mM KCl, 1 mM MgSO4) containing 38 mM ß-mercaptoethanol and 0.35 mg/ml X-Gal. The filters were incubated at 30°C for 12 d.
In vitro binding assay
For a typical binding experiment, 5075 OD units of yeast overexpressing GSTSec2p or GSTSec2 truncations were resuspended in lysis buffer containing PBS, 0.5% Triton X-100, 5 mM DTT, and protease inhibitors. Cells were disrupted in a bead beater using 0.5-mm zirconia/silica beads (beads and instrument from Biospec Products). Lysates were then cleared by centrifugation at 16,000 g for 10 min at 4°C. Triton X-100 was adjusted to 1% and supernatants were then incubated with 400 µl of a 50% (vol/vol) slurry of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) beads for 60 min while rotating the samples at 4°C. After the incubation, the beads were spun at 5,000 g and washed four times with 1 ml of ice cold PBS buffer. Hexa-histidinetagged (His6)Ypt32p and (His6)Sec4p were purified from Escherichia coli as previously described (Du and Novick, 2001). GSTSec2 fusion protein immobilized on glutathione-Sepharose beads (20 µl of beads; estimated amount of GSTSec2p on beads was 0.5 µg) was incubated with 3 nM Ypt32p in PBS buffer containing 1 mg/ml BSA, 1 mM DTT, and 1 mM MgCl2. Sec4p binding experiments were conducted in the same manner in PBS buffer containing 1 mg/ml BSA, 1 mM DTT, and 5 mM MgCl2. The total volume of incubation mixtures was 400 µl. After incubating for 60 min at room temperature, the resin was washed with the incubation buffer without BSA and bound products were separated by SDS-PAGE. To preload Ypt32p with GDP or GTP--S, 400 nM protein was incubated with 1 mM nucleotide in PBS buffer containing 1 mg/ml BSA, 1 mM EDTA, 1 mM MgCl2, and 1 mM DTT for 1 h at room temperature. Sec4p preloading buffer contained PBS, 1 mg/ml BSA, 5 mM MgCl2, and 1 mM DTT. To analyze the binding of the nucleotide-free forms of Ypt32p and Sec4p to GSTSec2p, the Rab proteins were incubated with GSTSec2p immobilized on beads in PBS buffer containing 5 mM EDTA, 1 mg/ml BSA, and 1 mM DTT. After the incubation, the beads were washed with PBS buffer containing 5 mM EDTA.
GDP displacement assay
GDP displacement activity was monitored as previously described (Walch-Solimena et al., 1997). Ypt32p (0.4 µM) was preloaded with [3H]GDP (0.8 µM; 27 Ci/mmol) by incubation in 50 mM Tris, pH 8, 100 mM KCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, and 1 mM DTT for 30 min at 30°C. After the incubation, MgCl2 was adjusted to 6 mM. Sec4p (0.4 µM) was preloaded with [3H]GDP (0.8 µM; 27 Ci/mmol) by incubation in a buffer containing 50 mM Tris, pH 8, 100 mM KCl, 1 mM EDTA, 6 mM MgCl2, 1 mg/ml BSA, and 1 mM DTT. Reactions were initiated by the addition of 0.5 mM GDP and either purified GSTSec2 protein or control buffer (total volume 30 µl) to 30 µl of [3H]GDP preloaded Ypt32p and Sec4p. To purify GSTSec2p, 1 liter of yeast (1.2 OD/ml) was grown in YPGal (2% galactose). Cells were resuspended in 10 ml of lysis buffer and lysed as described above. The lysate was centrifuged at 90,000 g for 45 min at 4°C. The supernatant was incubated with 3 ml of 50% slurry of glutathione-Sepharose 4B at 4°C for 60 min. After the incubation, the beads were spun and washed 3 times with 15 ml of ice cold PBS buffer. The bound protein was eluted with three 1-ml portions of elution buffer (50 mM Tris, pH 8, 10 mM glutathione). The eluted fractions were pooled, buffer exchanged, and concentrated using Ultrafree-4 centrifugation units (Millipore) with a 10,000 mol wt cut off. The purified protein was stored at -20°C in a buffer containing 20 mM Tris, pH 8, 100 µM PMSF, and 40% glycerol. GDP displacement from Ypt32p was conducted at 30°C. To lower the high intrinsic GDP release rate of Sec4p, experiments with Sec4p were conducted at 12°C. 10-µl aliquots were withdrawn at the times indicated and placed into 1 ml of ice cold stop buffer solution (25 mM Tris, pH 8, 20 mM MgCl2). Rab-associated radioactivity was determined by filter binding followed by scintillation counting.
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Footnotes |
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C. Walch-Solimena's present address is Max Planck Institute of Molecular Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
* Abreviations used in this paper: GAP, GTPase-activating proteins; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; ts, temperature sensitive; YPD, YP medium containing glucose; YPGal, YP medium containing galactose; YPGR, YP medium containing galactose and raffinose.
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Acknowledgments |
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This work was supported by a grant to P. Novick from the National Institutes of Health (CA46128).
Submitted: 2 January 2002
Revised: 9 April 2002
Accepted: 9 April 2002
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References |
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