Article |
Address correspondence to Vytas A. Bankaitis, Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. Tel.: (919) 962-9870. Fax: (919) 966-1856. E-mail: bktis{at}med.unc.edu
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Abstract |
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Key Words: Kes1p; phosphoinositides; yeast Golgi; Sec14p; ARF
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Introduction |
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Yeast express seven OSBPs. Three (Osh1p, Osh2p, and Osh3p) are classified as long OSBPs, typified by the mammalian OXYB, where a large extension is found NH2-terminal to the 430 amino acid oxysterol binding homology domain (Fig. 1 A). The remaining four are homologous to the oxysterol binding domain of OXYB and are classified as short OSBPs (Fig. 1 A). None of the yeast OSBPs individually plays an essential function, but deletion of all seven OSBP genes results in inviability (Jiang et al., 1994; Fang et al., 1996; Levine and Munro, 1998; Beh et al., 2001).
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Herein, we demonstrate that yeast Kes1p is a phosphoinositide (PIP) binding protein whose lipid binding activities are necessary, but not sufficient, for Kes1p localization to yeast Golgi membranes. We also show that Golgi complex localization is critical for Kes1p function. Finally, we present evidence to suggest that Kes1p activity influences ARF function in yeast. The collective data suggest that OSBPs are compartment specific regulators (or effectors) of ARF function in eukaryotes.
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Results |
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BLAST analyses also identify a motif (Kes1p residues 108149) that is present only in OSBPs and is highly conserved among them (Fig. 2 C). This OSBP domain consists of two 11-residue motifs separated by a linker of 20 divergent residues. Two of the kes1 alleles alter residues adjacent to (E107K), or within (H143Y), this domain.
Kes1p is a PIP binding protein
As PI(4,5)P2 and other PIPs are common ligands for PH domains, we assessed binding of purified Kes1p to PI(4,5)P2 or its soluble headgroup inositol-1,4,5-trisphosphate (IP3). To this end, [3H]-BZDC-PI(4,5)P2 and [3H]-BZDC-IP3 were used as photo-affinity ligands (Dorman and Prestwich, 1994; Prestwich, 1996; Prestwich et al., 1997; Chaudhary et al., 1998a,b; Feng et al., 2001). The [3H]-BZDC-PI(4,5)P2 binding experiments were performed in a mixed micelle system where the photo-affinity ligand was a trace component. Thus, the photolabeling assay requires Kes1p to interact with photo probe monomers in the face of large detergent excess. Although Sec14p fails to bind either probe (unpublished data), Kes1p binds both [3H]-BZDC-PI(4,5)P2 and [3H]-BZDC-IP3 avidly, as indicated by recovery of appropriate covalent adducts (Fig. 3 A). The intensities of Kes1p photolabeling were similar to those scored for the PIP binding protein gelsolin.
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To quantify Kes1p affinity for PI(4,5)P2, we determined the concentration of competitor required for 50% displacement of [3H]BZDC-PI(4,5)P2 from Kes1p (IC50). Although photoaffinity labeling is a nonequilibrium process (Dorman and Prestwich, 1994) and cannot directly give equilibrium dissociation constants, displacement by competing ligands yields a rank order of relative affinities. A dose-dependent reduction in photolabeling efficiency was observed when PI(4,5)P2 was employed as competitor (Fig. 3 B). The IC50 for PI(4,5)P2 is 1.9 µM with an estimated KD = 2.5 µM for Kes1p-[3H]BZDC-PI(4,5)P2 binding. These values resemble those measured for PI(4,5)P2 and [3H]BZDC-PI(4,5)P2 binding by PLC-1 (Tall et al., 1997; Lemmon, 1999).
Kes1p PH domain is sufficient for PIP binding
We tested whether the Kes1p PH domain represents the PI(4,5)P2 and IP3 binding module. Indeed, a 144 amino acid Kes1p fragment that consists largely of the PH domain (residues 171314) is sufficient for PI(4,5)P2 and IP3 binding (Fig. 3 C). A 110 amino acid region of Kes1p (residues 205314) that defines the putative Kes1p PH domain (Fig. 2 B) also binds IP3 photo-probe (unpublished data). Neither a smaller region of Kes1p (residues 205309) that fully overlaps this domain, nor a Kes1p domain (residues 220330) that incompletely overlaps this domain exhibits any inositide binding activity (unpublished data). Interestingly, the minimum Kes1p inositide binding module does not include W317, the signature residue of PH domains (Fig. 2 B; see below).
Specificity of PIP binding
To characterize specificity of Kes1p binding to PIPs, we employed a competitive displacement strategy using a variety of binding substrates in a Kes1p-[3H]BZDC-PI(4,5)P2 photolabeling assay. At 400-fold molar excess, all PIPs tested and other acidic phospholipids (phosphatidylserine, phosphatidic acid, and cardiolipin) displaced [3H]BZDC-PI(4,5)P2 (Fig. 3 D). Other lipids (phosphatidylethanolamine, phosphatidylcholine, ceramide, and diacylglycerol) were ineffective. Soluble inositol-polyphosphates (IP3, IP4, IP6) also failed to displace [3H]BZDC-PI(4,5)P2 from Kes1p (unpublished data). When unlabeled competitor lipids were reduced to a 10-fold molar excess relative to [3H]BZDC-PI(4,5)P2, only PI(4,5)P2 displaced photo-probe. PI, PI(4)P, phosphatidic acid (PA), and other acidic phospholipids were ineffective (unpublished data). A 10-fold molar excess of PI(3,4,5)P3, PI(3,4)P2, or PI(3,5)P2 also failed to displace [3H]BZDC-PI(4,5)P2 (Fig. 3 D). Thus, PI(4,5)P2 is the preferred Kes1p ligand in vitro.
Mutations in the Kes1p PH domain influence PIP binding
Since the Kes1p PH domain binds PI(4,5)P2, we investigated whether integrity of this domain is required for PIP binding. To this end, we characterized the binding properties of Kes1pE312K and Kes1p W317A (Kes1pW317A) using the photolabeling assay described above. In neither case could we detect binding of full-length mutant protein to [3H]BZDC-PI(4,5)P2 (Fig. 4 A) or to [3H]BZDC-IP3 (unpublished data). In vivo complementation experiments reveal that Kes1pE312K and Kes1pW317A, while stable proteins in yeast, are nonfunctional (unpublished data).
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The site of Kes1p proteolysis that generates Kes1pW317A* was determined. Matrix-assisted laser ionization coupled with time-of-flight mass analysis (MALDI-TOF) analyses yield a mass of 50.663 kD for Kes1p and 36.838 kD for Kes1pW317A* (Fig. 4 B). These data assigned the Kes1pW317A* cleavage site to the amide bond of a dibasic motif comprised of Kes1p residues R314 and K315, indicating that Kes1pW317A* is devoid of the COOH-terminal 120 residues (Fig. 4 B). Interestingly, this cleavage site lies upstream of the W317A substitution. Thus, Kes1pW317A* represents a proteolytic fragment consisting solely of wild-type Kes1p primary sequence.
To confirm this result, we purified GST-tagged versions of full-length Kes1p and of a protein fragment generated by engineering a Kes1p truncation immediately following residue R314. This truncation product is designated Kes1p(1314). Consistent with the Kes1pW317A* data, Kes1p(1314) binds [3H]BZDC-IP3 with an affinity that is similar to that exhibited by Kes1p itself (Fig. 4 C). We also tested whether the COOH-terminal truncation restores PIP binding to the mutant Kes1pE312K. This experiment was of interest for two reasons. First, it tests the allele specificity of the suppression effect elicited by the COOH-terminal truncation mutation. Second, because E312K is retained in the truncated product (unlike the W317A case where the fragment consists of wild-type Kes1p sequence), this experiment tests whether E312K imposes an intrinsic PIP binding defect on Kes1p. Photolabeling data demonstrated that Kes1pE312K* binds PIPs, whereas full-length Kes1pE312K cannot (Fig. 4 C), indicating deletion of the COOH-terminal 120 Kes1p residues rescues both Kes1pE312K and Kes1pW317A PIP binding defects. Thus, neither E312 nor W317 are intrinsically essential for PIP binding. Rather, COOH-terminal Kes1p sequences inhibit PIP binding by the Kes1p PH domain.
Kes1p mutants intrinsically defective in PIP binding are nonfunctional
The data indicate that Kes1pE312K and Kes1pW317A exhibit PIP binding defects of a regulatory nature. This complicates conclusions that can be drawn regarding the significance of PIP binding for Kes1p activity from functional analyses of Kes1pE312K and Kes1pW317A alone. To more directly assess the functional significance of PIP binding by Kes1p, we generated Kes1p derivatives with intrinsic defects in PIP binding. Our criteria for such mutants was that these will fail to bind inositide photo-probe, and that these binding failures will not be rescued by COOH-terminal Kes1p truncation.
To generate mutant Kes1p intrinsically defective in PIP binding, we were guided by structural analyses indicating that basic residues in the PH domain variable loop 1 and 2 regions engage inositide headgroups (Lemmon, 1999). We generated a triple mutant Kes1p (Kes1p3E) where residues R236, K242, and K243 in the presumed loop 2 region of the Kes1p PH domain were replaced with glutamate (see Fig. 2 B). Kes1p3E fails to bind photo-probe in vitro, and truncation of Kes1p3E distal to residue K314 fails to restore PIP binding (Fig. 4 D). Thus, Kes1p3E is intrinsically defective in PIP binding.
The biochemical PIP binding defect translates to loss of Kes1p activity in yeast. Complementation analyses demonstrate that Kes1p3E is nonfunctional in vivo (Fig. 4 D). This defect is manifested even though Kes1p3E is a stable cellular protein that accumulates to steady-state levels that are several-fold greater than those of wild-type Kes1p. Thus, PIP binding is required for Kes1p function in vivo.
Kes1p OSBP domain is nonessential for PIP binding
To investigate the OSBP motif in detail, we performed alanine-scanning mutagenesis throughout the OSBP domain (Fig. 5 A). Complementation experiments show that Kes1pHH143/144AA, Kes1pK109A, and Kes1pE117A are nonfunctional in vivo (Fig. 5 B), as are Kes1pLG115/116AA and Kes1pEQ139/140AA. All of these proteins are expressed as stable polypeptides in cells (unpublished data). Thus, the OSBP domain is critical for Kes1p function in yeast. Photolabeling experiments demonstrate that OSBP domain mutants avidly bind photo-probe in a manner that is subject to competitive displacement by unlabeled PI(4,5)P2 competitor (Fig. 5 A). These data show that the OSBP domain is not required for PI(4,5)P2 binding as scored by the photolabeling assay.
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Kes1p localizes to Golgi membranes
Fractionation studies revealed that Kes1p resides in both soluble and membrane-bound pools (Fang et al., 1996). To localize Kes1p more precisely, a YCp(KES1-YFP) plasmid was constructed that drives physiological expression levels of a chimera consisting of Kes1p fused via its COOH terminus to the green fluorescent protein (GFP) NH2 terminus. This chimera is both stable and functional (unpublished data). To assess Kes1p-GFP distribution, appropriate yeast strains were cultured to midlogarithmic growth phase in minimal medium at 26°C and cells were imaged. As control, we also monitored localization of GFP expressed from an isogenic YCp plasmid carrying a KES1 promoter cassette. Whereas GFP is distributed diffusely throughout the cytosol, Kes1p-GFP adopts both a diffuse cytosolic location and localization to 310 punctate structures dispersed throughout the cytoplasm (Fig. 6).
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Localization of Kes1p to Golgi membranes requires PIP binding
Previous reports had documented that PIP binding is necessary and sufficient for localization of long OSBPs to target membranes (Levine and Munro, 1998). To determine whether PIP binding plays the same role in targeting of short OSBPs, representative mutations in the Kes1p PH domain were introduced into a YCp(KES1-GFP) expression cassette. Localization of mutant Kes1-GFP proteins was then monitored in living cells by fluorescence microscopy.
As expected, the Kes1p3E-GFP chimera exhibit a diffuse intracellular distribution that contrasts with the punctate Golgi region staining recorded for wild-type Kes1p-GFP (Fig. 7 A). Thus, Kes1p3E-GFP fails to localize to Golgi membranes in vivo. We also assessed localization of Kes1pE312K and Kes1pW317A. These species fail to bind PIPs in vitro as full-length polypeptides, but regain PIP binding activity when the COOH-terminal 120 Kes1p residues are removed. Interestingly, Kes1pE312K-GFP and Kes1pW317A-GFP exhibit localization profiles similar to those recorded for wild-type Kes1p-GFP, suggesting Kes1pE312K and Kes1pW317A target to Golgi membranes (Fig. 7 A). Thus, unlike the PIP-binding defective Kes1p3E, Kes1pE312K and Kes1pW317A are apparently subject to regulatory compensation that exposes their PIP binding activities in the appropriate in vivo context. Kes1pE312K and Kes1pW317A remain nonfunctional, however.
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We also tested whether inactivation of the Golgi complexassociated Sec14p affects Kes1p localization. Sec14p stimulates PIP synthesis in a variety of in vitro systems (Hay and Martin, 1993; Cunningham et al., 1996; Jones et al., 1998). Moreover, Sec14p stimulates PI(4)P synthesis in yeast (Hama et al., 1999; Phillips et al., 1999). Kes1p localization is not compromised in sec14-1ts strains at 26°C. Shift of the sec14-1ts strain to 37°C for 30 min reduces, but does not abolish, Kes1p association with Golgi membranes (Fig. 7 B).
Localization of Kes1p to Golgi membranes requires a functional OSBP domain
Analyses of mutants compromised for OSBP domain function demonstrate that an intact OSBP domain is also essential for proper localization of Kes1p-GFP in living cells. Imaging experiments show that the Kes1pK109A-GFP chimera adopts a diffuse cytosolic distribution with no visible concentration in punctate structures (Fig. 7 C). Kes1p-GFP chimeras with mutant OSBP domains (i.e., Kes1pHH143/144AA-GFP, Kes1pLG115/116AA-GFP, and Kes1pVS141/142AA-GFP) also exhibit exclusively cytosolic staining profiles (unpublished data). Of interest is the allele specificity that underlies these localization defects. E107K represents a missense substitution at a position only two residues upstream of K109A. Yet, Kes1pE107K does not mistarget to the cytosol (unpublished data), whereas Kes1pK109A does. Finally, mutations that release Kes1p-GFP to the cytosol are not limited to the OSBP domain. For example, LH201/202FN also has this effect (unpublished data). We conclude the region bounded by Kes1p residues 109202 is critical for the Kes1p localization to membranes, and that the OSBP domain is an essential component of this membrane targeting information.
Because Kes1ps with defective OSBP domains bind PIPs, we tested whether this domain contributes to Golgi region targeting by binding another lipid. To this end, we determined whether the phospholipase D (PLD)-driven accumulation of PA that accompanies Sec14p inactivation is required for Kes1p localization to the yeast Golgi region. This effort was motivated by the weak binding of Kes1p to PA monomers (see above). PLD deficiency markedly reduces Golgi region/endosomal PA levels in Sec14p-deficient yeast (Li et al., 2000a), but has no effect on Kes1p localization in Sec14p-deficient strains (unpublished data). In accord with our previous findings that the involvement of Kes1p in yeast Golgi region function is independent of metabolic flux through the yeast sterol biosynthetic pathway (Fang et al., 1996), we also find that mutations compromising sterol biosynthesis (e.g., erg6) do not result in obvious mislocalization of Kes1p from Golgi membranes (unpublished data).
Relationship between magnitude of Kes1p overexpression required for compromise of "bypass Sec14p" and PI(4)P levels
Since genetic data indicate Kes1p antagonizes activity of the Sec14p pathway for Golgi region secretory function, we overexpressed Kes1p in "bypass Sec14p" mutant strains. In agreement with previous demonstrations (Fang et al., 1996), all mechanisms for "bypass Sec14p" are sensitive to increased KES1 gene dosage (Fig. 8 A). Kes1p is unique in this respect. Increased dosage of structural genes for enzymes of the CDP-choline pathway (i.e., CKI1, PCT1, and CPT1) or the Sac1p PIP phosphatase (SAC1) have no effect on nonallelic mechanisms for "bypass Sec14p" (unpublished data). Although modest overproduction of Kes1p compromises all other mechanisms of "bypass Sec14p", robust overexpression of Kes1p is required for this effect in sac1 mutants (Fig. 8 A).
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The reduction in PI(4)P recorded in the YEp(sac1TM) strain corrects the inositol auxotrophy and cs growth phenotypes of sac1 mutants, but fails to ablate sac1-mediated "bypass Sec14p" (unpublished data). However, Sac1p
TM-mediated reduction in PI(4)P levels strongly sensitizes the "bypass Sec14p" phenotype of sac1 mutants to elevated Kes1p. Whereas large increases in KES1 dosage are required for compromise of "bypass Sec14p" in sac1 mutants, modest increases in KES1 dosage ablate "bypass Sec14p" in sac1 YEp(sac1
TM) strains (Fig. 8 D). Thus, the magnitude of Kes1p overproduction required for compromise of "bypass Sec14p" is proportional to cellular PI(4)P levels.
Kes1p is mislocalized in sac1 mutants
Based on analyses of PI(4)P levels in sac1 mutants bearing pik1ts and stt4ts mutations, it is clear that the accumulated PI(4)P is predominantly synthesized via the Stt4p PI 4-kinase and not the Pik1p kinase (Nemoto et al., 2000; Foti et al., 2001). Because Pik1p is the Golgi regionlocalized PI 4-kinase (Walch-Solimena and Novick, 1999) and PI(4)P is likely the physiological ligand for Kes1p, we assessed the localization of Kes1p-GFP in sac1 mutants. As shown in Fig. 9 A, the intracellular profile of Kes1p-GFP distribution in sac1 mutants is dramatically different from its normal punctate distribution in wild-type strains. Rather, Kes1p-GFP localizes to a few large patches in sac1 cells that are frequently located in a juxtanuclear position. This altered Kes1p localization pattern does not reflect abnormal sac1 Golgi region morphology. Distribution of the Golgi complex marker Kex2p is not altered in sac1 mutants (Fig. 9 B). Rather, consistent with the significant (if not exclusive) localization of a Sac1p to ER compartments in yeast and mammalian cells (Whitters et al., 1993; Nemoto et al., 2000; Foti et al., 2001), we conclude that Kes1p is mistargeted to what are likely ER membranes in sac1 mutants. We suggest that Kes1p is mistargeted to ER membrane domains enriched in the PI(4)P that accumulates in these mutants. In support of this idea, the Kes1p PH domain is required for this mislocalization as Kes1p3E-GFP retains its cytosolic localization in sac1 mutants, and fusion of the Sac1p PIP phosphatase catalytic domain to the ER-resident protein Sec61p yields a fully functional Sac1p whose expression complements all sac1 phenotypes (including the "bypass Sec14p" phenotype [unpublished data]).
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Genetic interactions of kes1 with ARF and ARFGAP deficiencies
Kes1p deficiency elicits a "bypass Sec14p" that, like all "bypass Sec14p" phenotypes, requires Gcs1p ARFGAP activity (Yanagisawa, L., and V.A. Bankaitus, unpublished data). Our finding that Kes1p overproduction mimics Gcs1p defects in "bypass Sec14p" mutants further supports the possibility that Kes1p influences regulation of the ARF cycle. Therefore, we tested whether kes1 suppresses phenotypes associated with Gcs1p or ARF dysfunction.
Sodium fluoride (NaF) sensitivity is associated with defects in the yeast ARF cycle (Zhang et al., 1998). Gcs1p-deficient mutants are sensitive to NaF, as are arf2 strains carrying the arf1-3ts mutation (Zhang et al., 1998; Table I). Based on the results described above, we predicted that Kes1p defects might suppress NaF sensitivity in gcs1
and arf1-3 arf2
strains. Although both gcs1
and arf1-3 arf2
mutants fail to grow in the presence of 30 and 50 mM NaF, isogenic wild-type strains are NaF resistant (Table I). However, kes1
suppresses Gcs1p and ARF1p deficiency in this assay as isogenic kes1
gcs1
and kes1
arf1-3 arf2
mutants grow when challenged with either 30 or 50 mM NaF (Table I). Serial dilution experiments indicate that the viability of gcs1
strains is increased
1,000-fold relative by kes1
.
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Discussion |
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The PIP binding activity resides in what appears to be a Kes1p PH domain. Although the existence of PH domains has been documented for long OSBPs (e.g., OXYB yeast Osh1; Levine and Munro, 1998), our findings represent the first demonstration that short OSBPs also binds PIPs. Moreover, our data demonstrate that PIP binding, and its role in targeting an OSBP to Golgi membranes, is a functionally important activity in cells. With regard to the binding of Kes1p to other lipids, we failed to detect significant binding of Kes1p to 25-hydroxycholesterol (unpublished data).
One unanticipated result obtained from analysis of mutations in the Kes1p PH domain is our finding that the E312K and W317A mutations abolish PIP binding in the context of full-length Kes1p, but not when the COOH-terminal 120 Kes1p residues are removed. Thus, E312K and W317A do not affect PIP binding directly. Rather, these substitutions likely affect Kes1p conformation in such a way that the PH domain is not accessible to PIPs. Consistent with this model, E312 and W317 do not reside in the predicted PIP binding loops of the Kes1p PH domain, and truncation of the Kes1p COOH terminus does not rescue PIP binding defects associated with mutations in those loops. Because removal of the Kes1p COOH terminus suppresses E312K- and W317A-associated PIP binding defects, we speculate that the Kes1p COOH terminus interferes with PH domain function by occluding the PIP binding site. This proposal suggests that PIP binding by Kes1p is subject to conformational regulation by posttranslational modification, or by interaction of Kes1p with some other binding partner (either protein or lipid). This concept is supported by our demonstration that the E312K and W317A substitutions do not abrogate Kes1p targeting to the Golgi complex in vivo, whereas intrinsic Kes1p defects in PIP binding and defects in PIP synthesis by the Pik1p PI-kinase do.
Unlike the case for long OSBPs, PIP binding is insufficient for Kes1p targeting to Golgi membranes. An intact OSBP domain is also required. These data provide the first indication of a function for this signature motif of OSBPs. It remains unclear how the OSBP domain is involved in localization of Kes1p to Golgi membranes. One possibility is that the Kes1p OSBP motif interacts with the Kes1p COOH terminus to expose the PH domain for PIP binding. We do not find compelling evidence in favor of this model. Inactivating mutations in the OSBP domain neither abolish PIP binding by Kes1p, nor do these strongly compromise the affinity of Kes1p binding of PIP monomers in a mixed micelle system. Alternatively, the OSBP domain may increase the avidity of Kes1p for Golgi membranes by binding another Golgi complexlocalized ligand. Finally, the OSBP domain may represent an interface for homotypic binding of Kes1p molecules to each other so that the PH domains are configured in a dimeric arrangement where the PIP binding loops of each PH domain unit are exposed on one face of the dimer. This would generate a PIP binding module with a higher affinity for PIP than that exhibited by monomeric Kes1p. However, an increased binding affinity of dimeric Kes1p would not be apparent in the mixed micelle system we have employed to measure Kes1pPIP interaction.
Although Kes1p plays an important role in Golgi function in yeast (Fang et al., 1996), the in vivo functions of OSBPs in eukaryotic cells remain unresolved. We suggest that Kes1p function somehow interfaces with activity of the yeast ARF cycle. We find that arf1 and gcs1
both abrogate kes1
-mediated "bypass Sec14p", and several other lines of genetic evidence indicate that Kes1p dysfunction mimics elevated ARFGAP function, whereas elevated Kes1p phenocopies reduced ARFGAP function. Thus, Kes1p may act as: (a) an inhibitor of the Gcs1p ARFGAP activity that is required for yeast Sec14p-dependent Golgi complex function (Yanagisawa, L., and V.A. Bankaitus, unpublished data), (b) a novel ARF nucleotide exchange factor, or (c) an activator of an ARF nucleotide exchange factor. In our hands, Kes1p fails to inhibit the ARFGAP activity of Gcs1p in vitro and we have not detected intrinsic ARF nucleotide exchange activity associated with Kes1p (unpublished data).
Our data suggest that Kes1p may regulate ARF function through its effects on PIP synthesis via the Golgi complexassociated Pik1p as evidenced by the demonstration Kes1p defects partially suppress growth defects associated with Pik1p dysfunction. Kes1p may function, directly or indirectly, as a Pik1p inhibitor in vivo. Alternatively, effects of Kes1p on the ARF cycle may influence Pik1p activity. Linkage of Kes1p localization and function to Pik1p-mediated PI(4)P synthesis suggests that PI(4)P is the physiological Kes1p ligand. Although PI(4,5)P2 is bound by Kes1p with a 10-fold higher affinity, PI(4)P mass is fourfold greater than that of PI(4,5)P2 in yeast. Moreover, defects in PI(4,5)P2 synthesis do not compromise Kes1p localization to Golgi membranes, and massive accumulation of PI(4)P in a non-Golgi compartment entices Kes1p from Golgi membranes. The latter result suggests that a component of the mechanism by which sac1-mediated PI(4)P accumulation contributes to "bypass Sec14p" is an indirect one that involves Kes1p mistargeting to what is likely the ER. Finally, the data identify Kes1p as a reporter for Pik1p-driven PIP synthesis on yeast Golgi membranes. Thus, Pik1p facilitates the paradoxical recruitment to Golgi membranes of a protein whose dysfunction permits Sec14p-independent secretory function of Golgi membranes.
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Materials and methods |
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Site-directed mutagenesis
Mutageneses employed QuickChangeTM (Stratagene) and confirmed by DNA sequencing analysis (Sanger et al., 1977) using the Sequenase version 2.0 kit (Amersham Pharmacia Biotech).
Plasmid construction
Details of the various plasmid constructions are included in supplemental materials or are available from the authors by request.
Expression and purification of His6-tagged and GST-tagged proteins from E. coli
One liter of Superbroth (12 g tryptone, 24 g yeast extract, 4 g glycerol, 0.17 M KH2PO4, 0.72 M K2HPO4, 50 µg/ml ampicillin) was inoculated with 10 ml of an overnight culture of E. coli strain KK2186 harboring plasmids expressing His6- or GST-tagged proteins. Protein production was induced with isopropyl ß-D-thiogalactopyranoside (0.5 mM) and, after 35 h, cells were harvested by centrifugation. Cells were disrupted by sonication, and Triton X-100 was added (final concentration 0.1% vol/vol). After addition of DNase I (10 µg/ml) and MgCl2 (10 mM), lysate was clarified and filtered.
His6-tagged proteins were purified by HiTrap Ni-chelating column chromatography (Amersham Pharmacia Biotech) as per the manufacturer's instructions.
Peptide sequencing
Purified proteins were transferred to Immuno-Blot PVDF membranes (Bio-Rad Laboratories) using the semidry Transblot apparatus (Bio-Rad Laboratories). Blotted PVDF membrane were washed with distilled water and stained with Ponceau S. Kes1p species were excised from membranes and subjected to automated NH2-terminal Edman degradation.
Mass spectrometry
MALDI-TOF mass spectrometry was performed using positive mode on a Voyager Elite unit with delayed extraction technology (PerSeptive Biosystems). Samples were diluted 1:10 with matrix, and 1 µl of the resulting mix was deposited onto a smooth plate. Acceleration voltage was set at 25 kV and 1050 laser shots were summed. Sinapinic acid (D13, 460-0; Sigma-Aldrich) dissolved in acetonitrile: 0.1% TFA (1:1) was used as matrix. The mass spectrometer was calibrated with bovine serum albumin.
Online supplemental materials
PIP photolabeling assay. Photolabeling and displacement assays were performed as described previously (Kearns et al., 1998b). Details are available at http://www.jcb.org/cgi/content/full/jcb.200201037/DC1 or from the authors by request.
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Footnotes |
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* Abbreviations used in paper: ARF, adenosine diphosphate-ribosylation factor; ARFGAP, ARF guanosine triphosphatase activating protein; BZDC, ([p-benzoyldihydrocinnamidyl]-amino)propyl; GFP, green fluorescent protein; IP3, inositol-1,4,5-trisphosphate; MALDI-TOF, matrix-assisted laser ionization coupled with time-of-flight mass analysis; OSBP, oxysterol binding protein; PA, phosphatidic acid; PC, phosphatidylcholine; PH, Pleckstrin homology, PI, phosphatidylinositol; PIP, phosphoinositide; PLD, phospholipase D.
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Acknowledgments |
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Submitted: 8 January 2002
Revised: 14 February 2002
Accepted: 14 February 2002
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References |
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