Domain Analysis and Rational Mutant Design
The Fab1 lipid kinase is an ~250-kDa protein made up of several domains (A; Efe et al., 2005
; Michell et al., 2006
). To further understand how these domains contribute to the function of Fab1, we adopted a structural-genetic approach based on rational mutant design and assessed PtdIns(3,5)P2
levels, growth, and vacuolar morphology.
Figure 1. Characterization of Fab1 point mutants. (A) Schematic representation of the Fab1 domain structure depicting the FYVE domain, the CCT, the CCR domains that together form the GroL region, and the lipid kinase domain. Mutations or truncations were introduced (more ...)
To perturb the C-terminal lipid kinase domain (KIN), we truncated the entire kinase domain from residues 1652 to 2279 (Fab1ΔKIN
encoded by pRB415A::Fab1ΔKIN
) or used the Fab1D2134R
], a previously characterized mutation that ablates lipid kinase activity (A; Gary et al., 1998
). The FYVE domain was disrupted by eliminating the first 676 amino acids of Fab1 (Fab1ΔHindIII
; encoded by pRB415A::Fab1ΔHINDIII
) or more conservatively, by mutating cysteine262
]. This cysteine is part of a basic cluster found in all FYVE domains that maintains structural integrity by coordinating a Zn2+
ion (Stenmark et al., 1996
; Burd and Emr, 1998
The remaining region between residues 800 and 1500 contains the CCT and CCR domains (Efe et al., 2005
; Michell et al., 2006
). Residues ~800 to ~1050 are related to the CCT chaperonin family, whose members catalyze folding of actin and tubulin (Michell et al., 2006
). Residues ~1100–1500 contain multiple conserved cysteine and histidine residues and have been referred to as the CCR domain or as the PIPKIII-unique domain (Efe et al., 2005
; Michell et al., 2006
). Sequence alignment using the conserved domain database places the CCT and CCR domains within the GroL superfamily. Therefore, we refer to the combined CCT and CCR domains as the GroL-related region. To help delineate the function of these two domains, we mutated a conserved motif in the CCT region (T1017
ILLR to I1017
) and cysteine1243
in the CCR domain (Fab1CCR(C/A)
; , A and B).
Effect of fab1 Mutations on Incorporation of [3H]Inositol into PtdIns(3,5)P2 and Vacuole Morphology
We first used HPLC to measure levels of [3H]inositol-labeled PtdIns(3,5)P2 [3H]PtdIns(3,5)P2] in fab1Δ cells expressing the above-mentioned alleles of FAB1 (C). Cells expressing wild-type Fab1 from a CEN-based vector displayed [3H]PtdIns(3,5)P2 levels comparable with cells with an intact chromosomal copy of FAB1, whereas [3H]PtdIns(3,5)P2 was virtually undetectable in cells expressing Fab1KIN(D/R) (0.11 ± 0.01 vs. 0%; C). In contrast, expression of Fab1FYVE(C/S), Fab1CCT(T/I), or Fab1CCR(C/A) produced intermediate levels of [3H]PtdIns(3,5)P2. Fab1CCR(C/A) showed the greatest decline in lipid levels [0.024 ± 0.01 or 22% of wild-type [3H]PtdIns(3,5)P2], followed by Fab1CCT(T/I) [0.046 ± 0.01 or 42% of wild-type [3H]PtdIns(3,5)P2] and then by the FYVE point mutant [0.07 ± 0.01 or 63% of wild-type [3H]PtdIns(3,5)P2, C]. These data suggest that the Fab1 kinase is more sensitive to perturbations in the CCT and CCR regions compared with its FYVE domain, likely because the GroL-like region may regulate Fab1 kinase activity.
We next investigated whether the fab1
mutants in question could support growth under heat stress (D). fab1
Δ cells are incapable of growing at 38°C and exhibit a growth rate that is 2.5 times slower than wild-type cells at 26°C in rich medium (Yamamoto et al., 1995
). Consistent with this, fab1
Δ cells expressing Fab1KIN(D/R)
grew slowly at 26°C and were growth deficient at 38°C (D). This was in contrast to cells expressing wild-type Fab1 and Fab1FYVE(C/S)
, which grew comparably well at 26 and at 38°C (D). fab1
Δ cells expressing Fab1ΔHindIII
also grew similarly well at 38°C, indicating that Fab1FYVE(C/S)
is not a hypomorph of the FYVE domain (Supplemental Figure S1A). However, alteration of the GroL-related sequence affected growth under heat stress, whereas expression of both Fab1CCT(T/I)
ameliorated the growth of fab1
Δ cells at 26°C, these mutant alleles were defective for growth at 38°C (D).
Last, we explored the effect of the listed FAB1
alleles on vacuolar morphology (E). Elimination of PtdIns(3,5)P2
by deletion of FAB1
or by inactivation of its kinase activity, leads to a conspicuously enlarged, single-lobed vacuole (E; Yamamoto et al., 1995
; Gary et al., 1998
). Similarly, expression of Fab1CCR(C/A)
did not rescue vacuolar morphology in a fab1
Δ strain (E). We also compared mutations in cysteine1247
, and cysteine1292
and found they all caused single-lobed, enlarged vacuoles in cells (data not shown). This implies that the cysteines in the CCR region participate in a crucial but uncharacterized function that modulates Fab1 kinase activity. By comparison, expression of Fab1CCT(T/I)
mutant alleles resulted in vacuoles that were predominantly single-lobed but near wild-type size (E), likely because sufficient PtdIns(3,5)P2
was maintained in these mutants.
These data indicate that the FYVE domain is largely dispensable for the synthesis of PtdIns(3,5)P2. Mutating the FYVE domain of Fab1 only abated the 3H-labeled lipid amount by ~40%, and the cells exhibited near-normal vacuolar morphology and survived during heat stress (). In comparison, perturbing the CCT and CCR regions produced more deleterious effects on the Fab1 lipid kinase activity; consequently, cells displayed larger vacuoles and/or were temperature sensitive for growth (). This indicates that the CCT and CCR domains are important regulators of the Fab1 kinase, despite the possibility that the applied mutations did not fully incapacitate these domains.
Characterization of the Fab1 domain point mutants
Effect of fab1 Mutations on Membrane Association
To determine the structural-genetic requirements for Fab1 localization to the vacuole, we visualized GFP-fusion proteins of Fab1 and of its mutant alleles (). GFP fusion of wild-type Fab1 rescued vacuolar morphology (Supplemental Figure S1B) and PtdIns(3,5)P2
levels (data not shown). However, to avoid “dilution” of the fluorescence signal due to vacuole enlargement when expressing certain Fab1 mutants, we expressed GFP-fusion constructs at endogenous levels in wild-type yeast cells. Similar observations were obtained when these GFP-fusion proteins were expressed in a fab1
Δ strain (Supplemental Figure S1B). The vacuolar limiting membrane was labeled with the FM4-64 dye, and quantification of the fluorescence intensity was done as described previously (, A and B; Efe et al., 2007
). We used two methods to analyze the behavior of the GFP-fusion proteins: 1) fluorescence line plotting to determine fluorescence correlation between GFP and FM4-64 signal (A) and 2) quantification of the vacuole-to-cytosol (V/C) fluorescence ratio of the GFP signal (B).
Figure 2. Localization of Fab1 mutants. (A) Wild-type 6210 cells or vps34Δ cells expressing GFP-tagged Fab1 and its mutant forms at endogenous protein levels. Vacuoles were labeled with FM4-64. FM4-64 and DIC images were merged. Line plot fluorescence measurement (more ...)
Consistent with Fab1 enrichment on the vacuolar membrane relative to the cytosol, the fluorescence intensity peak of Fab1-GFP correlated well with FM4-64 and exhibited a V/C = 1.78 ± 0.28 (n = 25; , A and B). The V/C ratios for Fab1CCR(C/A)-GFP and Fab1KIN(D/R)-GFP were indistinguishable from the wild-type counterpart (1.88 ± 0.34, n = 27 and 1.71 ± 0.19, n = 28, respectively), suggesting that the kinase activity and the CCR region do not impact membrane association (B). Mutation of other cysteines in the CCR region did not hinder localization of the lipid kinase as well (data not shown). However, although Fab1CCT(T/I)-GFP bound to the vacuolar membrane, the V/C ratio of this mutant was reduced compared with Fab1-GFP (1.47 ± 0.18; n = 23 vs. 1.78 ± 0.28; p <0.05), implying that the CCT region may have a role in stabilizing Fab1 on the vacuole. We also determined that more drastic alterations to Fab1, such as truncating the CCT domain (GFP-Fab1CCTΔ) or the kinase domain (GFP-Fab1KINΔ), still allowed for at least partial association with the vacuole (Supplemental Figure S2).
In striking contrast, Fab1FYVE(C/S)-GFP and GFP-Fab1ΔHindIII were both displaced to the cytosol (A and Supplemental Figure S2). This is corroborated by a low V/C ratio of 1.07 ± 0.08 for Fab1FYVE(C/S)-GFP (B; n = 24). Moreover, Fab1-GFP was completely cytosolic when expressed in vps34Δ cells (A), suggesting that the FYVE domain and PtdIns(3)P are required for Fab1 association with the vacuole. Indeed, the FYVE domain of Fab1 (FYVEFab1) was sufficient for localization to the vacuole (A). Its distribution was dissimilar from the endosome-specific FYVE domain of EEA1 (FYVEEEA1) coexpressed in the same cells (A). Despite this difference in distribution, they were both cytosolic in vps34Δ cells (A).
Behavior of the FYVE domain of Fab1. (A) Wild-type (top) or vps34Δ cells (bottom) coexpressing GFP-FYVEFab1 and RFP-FYVEEEA1. (B) fab1Δ (top) or vac7Δ (bottom) cells expressing GFP-FYVEFab1. Bar, 5 μm.
To the best of our knowledge, no reports exist suggesting a lipid partner for the FYVE domain other than PtdIns(3)P. Nonetheless, we postulated that the vacuolar distribution of the FYVEFab1 might occur by binding to PtdIns(3,5)P2, which is also eliminated in vps34Δ cells, or by binding to a vacuolar specific protein partner such as Vac7. Notably, the FYVEFab1 remained bound to the vacuole when expressed in fab1Δ or vac7Δ cells, excluding PtdIns(3,5)P2 and Vac7 as determinants of vacuolar localization (B). In comparison, GFP fusion of the GroL-like region did not seem to associate with the vacuole and was predominantly cytosolic (Supplemental Figure S2).
Together, these observations suggest that the FYVE domain is the primary membrane targeting mechanism for Fab1 but that the CCT domain may play a role in stabilizing this association. Thus, it is possible that Fab1FYVE(C/S) might retain weak and transient association with the vacuole through a CCT-mediated interaction, explaining its ability to complement fab1Δ cells.
The Fab1 Kinase Recruits Vac14 to the Vacuole
Rudge et al. (2004)
previously showed that Vac14 and Fig4 mislocalize to the cytosol in fab1
Δ cells. The authors suggested that this was an indirect effect because they could not detect an interaction between Fab1 and Vac14/Fig4. Never-theless, it remained possible that Fab1 targets Vac14 and Fig4 to the vacuole through protein–protein interactions, or indirectly, through the synthesis of PtdIns(3,5)P2
or even through protein kinase activity. To distinguish between a role for the Fab1 protein versus its catalytic activity, we used the kinase-dead Fab1 mutant Fab1KIN(D/R)
(Gary et al., 1998
). We labeled the vacuolar membrane with FM4-64 and acquired paired images of Vac14-GFP and FM4-64 by epifluorescence microscopy and quantified the V/C fluorescence ratio for Vac14-GFP (). Vac14 function is not significantly affected by GFP fusion (Rudge et al., 2004
Figure 4. Localization of Vac14-GFP in cells expressing fab1 mutants. (A) fab1Δ VAC14-GFP cells transformed with vector, FAB1 or its mutants. Cells were labeled with FM4-64 to denote the vacuolar membrane. FM4-64 is shown overlapped with the corresponding (more ...)
As expected, the fluorescence peaks of Vac14-GFP and FM4-64 coincided well in FAB1 cells, but it failed to do so in fab1Δ cells (A). The V/C ratio for Vac14-GFP in FAB1 cells (V/CFAB1) was 2.16 ± 0.55 (n = 20) versus 0.96 ± 0.2 (n = 20) in fab1Δ cells (V/Cfab1Δ; B). In comparison, cells expressing fab1KIN(D/R) not only exhibited enlarged vacuoles but also the fluorescence profiles for Vac14-GFP and FM4-64 correlated well. Furthermore, the V/C ratio for Vac14-GFP in these cells was statistically indistinguishable from V/CFAB1 (1.88 ± 0.43, n = 21; p > 0.05; ). This indicates that Fab1 acts structurally to localize Vac14 to the vacuole and independently of its kinase activity or PtdIns(3,5)P2. Corroborating this conclusion, Vac14-GFP was displaced from the vacuole in cells expressing the FYVE-point mutant of Fab1, which itself is mislocalized to the cytosol (, A and B). The V/C ratio for Vac14-GFP in fab1Δ cells and those expressing Fab1FYVE(C/S) was statistically the same (1.07 ± 0.18; n = 20 vs. 0.96 ± 0.2; p > 0.05).
We also explored the behavior of Vac14-GFP in cells expressing the Fab1CCT(T/I) and Fab1CCR(C/A) point mutants. The V/C ratio of Vac14-GFP in cells expressing Fab1CCT(T/I) or Fab1CCR(C/A) was 1.21 ± 0.13 (n = 20) and 1.19 ± 0.2 (n = 20), respectively, which was significantly less than wild-type V/C. Thus, perturbing the CCT and CCR domains of Fab1 led to a measurable dissociation of Vac14-GFP from the vacuole (). However, it is important to point out that Vac14-GFP maintained partial association with the vacuole in the GroL-like domain mutants because their V/C ratios were statistically higher than V/Cfab1Δ (B). This is consistent with the possible hypomorphic nature of these mutants and/or the presence of an additional site on Fab1 may contribute to localization of Vac14 (). The localization of Fig4-GFP was similar to that of Vac14-GFP; expression of Fab1FYVE(C/S), Fab1CCT(T/I) and Fab1CCR(C/A) in fab1Δ cells did not rescue Fig4-GFP localization to the vacuole, whereas expression of Fab1 or Fab1KIN(D/R) did (Supplemental Figure S3). Together, these data suggest that Fab1 has a direct role in targeting Vac14 and Fig4 to the vacuole, perhaps via interactions with the CCT and CCR domains.
The Fab1 Kinase Forms a Vacuolar Protein Complex with Vac14 and the Fig4 Phosphatase
Our observations indicated that Fab1, Vac14, and Fig4 might assemble into a single protein complex in yeast cells. It was shown previously that Vac14 and Fig4 coIP and are mutually dependent for association with the vacuolar membrane (Rudge et al., 2004
; Duex et al., 2006b
). Indeed, we observed coIP of Vac14-HA and Fig4 (A). Nevertheless, previous attempts to demonstrate an interaction between Fab1 and Vac14 (or Fig4) were unsuccessful (Bonangelino et al., 2002
; Rudge et al., 2004
). However, we now demonstrate that Myc-tagged Fab1 was recovered with Vac14-HA (A). Most notably, IP of Fab1-Myc retrieved not only Vac14-HA but also the Fig4 phosphatase as well (A). Although it is possible that all three proteins interact with each other in pairwise combinations, we interpret these results as support for a protein complex that contains all three proteins together.
Figure 5. Fab1, Vac14 and Fig4 interact with each other. Monoclonal anti-HA or anti-Myc antibodies were used for IP from solubilized whole cell lysates (A and D) or from cytosolic/membrane fractions (C) as described in Materials and Methods. IPs were separated (more ...)
We then tested whether this putative Fab1 complex could be detected in both membrane and cytosolic fractions. We first fractionated whole cell lysates into cytosolic and membrane fractions and observed a relative distribution for Fab1-Myc, Vac14-HA, and Fig4 of ~70% versus ~30% between the membrane and cytosolic fractions, respectively (B). The absence of the Vph1 vacuole membrane marker from the cytosolic fraction indicates the absence of contaminating membrane (B). Additionally, this distribution was comparable with the relative distribution of GFP-fusion proteins by microscopy. We observed that Fab1-Myc and Fig4 were both recovered with Vac14-HA from the membrane fraction (C). In contrast, IP of Vac14-HA from the cytosolic fraction recovered Fig4, but not Fab1-Myc (C). These data indicate that Vac14 and Fig4 can interact in the cytosol but that association of Fab1 with Vac14 and Fig4 is stimulated and/or stabilized on the vacuolar membrane.
We also tested whether Fab1, Fig4 and Vac14 existed as monomers or whether they possibly displayed higher order organization. To test this, we simultaneously expressed in the same yeast cells two alleles of FAB1
, and VAC14
encoding two distinct epitopes (D). We did not observe coIP between Fig4-Myc and Fig4-HA not between Fab1-Myc and Fab1-HA (D). In contrast, Vac14-FLAG was retrieved with HA-Vac14 by IP (D), which is consistent with yeast two-hybrid assays showing Vac14–Vac14 interaction (not illustrated; Dove et al., 2002
). Overall, these results indicate that Fab1, Vac14, and Fig4 can physically associate to form a membrane-bound protein assembly. Likely, there are multiple copies of Vac14 per complex, whereas the Fab1 kinase and the Fig4 phosphatase may be present at one subunit per complex.
Vac14 and Fig4 Exhibit Mutually Dependent Interaction with the Fab1 Kinase
To better understand how the Fab1 complex forms, we performed coIP experiments in different mutant backgrounds. We first determined that Vac14 and Fig4 bound to each other in the absence of the Fab1 kinase (A). This is consistent with a putative Vac14–Fig4 subcomplex observed in the cytosolic fraction (C). We also established that the Fab1 kinase and the Fig4 phosphatase were not retrieved together from vac14Δ cell lysates, which supports a role for Vac14 as a linker between the kinase and the phosphatase (A). Remarkably though, Fab1 and Vac14 interacted poorly in the absence of Fig4 (A), which suggests that Fig4 may play a regulatory/structural role in the assembly of the Fab1 complex.
Figure 6. Vac14 and Fig4 are both required for interaction with Fab1. (A) IPs were done with monoclonal anti-HA or anti-Myc from whole cell lysates from fab1Δ cells with or without HA-tagged Vac14 (top left), from vac14Δ cells with or without Myc-tagged (more ...)
Deletion of VAC7
leads to undetectable levels of PtdIns(3,5)P2
(Bonangelino et al., 1997
; Gary et al., 2002
). Because the mechanism for this regulation remains unknown, we postulated that Vac7 might be important for the association of Fab1 with Vac14-Fig4. However, deleting VAC7
did not prevent Fab1 from interacting with Fig4, seemingly excluding this hypothesis (A). Conversely, hyperosmotic shock of yeast cells causes a dramatic increase in PtdIns(3,5)P2
levels after 5–10 min, followed by an abatement after 20–30 min (Dove et al., 1997
; Duex et al., 2006b
). Nonetheless, changes in PtdIns(3,5)P2
did not seem to correlate with changes in the amount of Fab1 complex; we did not observe a significant change in the levels of Fig4 coIPed with Fab1-Myc upon exposure to salt-shock for 0, 8, or 25 min (Supplemental Figure S4). Together, these results indicate that Vac14 and Fig4 are mutually dependent for interaction with Fab1 and may explain the concomitant role of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P2
(Duex et al., 2006a
; Efe et al., 2007
Fig4, but Not Its Phosphatase Activity, Is Required for Atg18-dependent Hyperactivation of the Fab1 Kinase
Atg18 was identified as an effector of PtdIns(3,5)P2
that regulates vacuolar morphology and membrane recycling. Atg18 also regulates PtdIns(3,5)P2
levels—deletion of Atg18 leads to an approximately ninefold increase in PtdIns(3,5)P2
levels (Dove et al., 2004
). We showed previously that this increase is dependent on FIG4 (Efe et al., 2007
). Additionally, Fig4 is required to increase PtdIns(3,5)P2
levels during hypertonic shock (Duex et al., 2006a
). This unexpected role for Fig4 in the synthesis of PtdIns(3,5)P2
is likely explained by our observations—Fig4 is necessary for formation and/or stability of a productive Fab1-Vac14 interaction.
Conceivably, Fig4 may stimulate PtdIns(3,5)P2
synthesis by mediating direct protein–protein interactions or through its phosphatase activity, possibly by acting as a protein phosphatase (Duex et al., 2006b
). To differentiate between these possibilities, we used a catalytic-inactive FIG4
, which was initially isolated as a suppressor of the vac7
Δ phenotype (Gary et al., 2002
; Rudge et al., 2004
The basal level of [3H]PtdIns(3,5)P2 in atg18Δ fig4Δ cells was 0.13 ± 0.03% of total [3H]PtdIns, which is similar to wild-type [3H]PtdIns(3,5)P2 levels (0.11%; B). When Atg18 was expressed episomally—recreating the fig4Δ condition—[3H]PtdIns(3,5)P2 levels were slightly decreased (0.07 ± 0.01%). By contrast, expression of wild-type FIG4 in atg18Δ fig4Δ cells recapitulated [3H]PtdIns(3,5)P2 levels in atg18Δ cells (0.81 ± 0.05 vs. 0.9%; B). Similarly, expression of fig4-1 in atg18Δfig4Δ cells produced even higher levels of [3H]PtdIns(3,5)P2 (1.37 ± 0.11% of total PtdIns; B), likely because of abated turnover of the lipid. These results support the notion that Fig4 provides structural stability to the Fab1–Vac14 interaction independently of its phosphatase activity. To confirm this notion, we tested whether the Fab1 complex was reconstructed in atg18Δ fig4Δ cells expressing vector, FIG4 or fig4-1. As shown in A, the absence of Fig4 prevented coIP between Fab1-Myc and Vac14-HA (C). In contrast, expression of FIG4 or of the phosphatase-dead fig4-1 allele permitted corecovery of Fab1-Myc and Vac14-HA (C). Therefore, these observations support a structural role for Fig4 in stabilizing a productive Fab1 complex.
The GroL-like Domain of Fab1 Is Required for Interaction with Vac14 and Fig4 in Cells
Expression of Fab1CCT(T/I) or of Fab1CCR(C/A) crippled vacuole association of Vac14 and Fig4 ( and Supplemental Figure S3). This suggests that the GroL-like region of Fab1 might be a binding site for Vac14 and/or Fig4. To test this hypothesis, we expressed a HA-tagged GroL-like fragment in fab1Δ cells and assessed the recovery of FLAG-tagged Vac14 and Fig4 by coIP. As shown, both Vac14-FLAG and Fig4 were recovered with the HA-GroL domain but not from control cells expressing the empty vector (A). Importantly, mutating the TILLR motif in the CCT domain to ILLLA blocked interaction with Vac14 and Fig4, which is congruous with the release of Vac14 and Fig4 from the vacuole membrane in cells expressing Fab1CCT(T/I) ( and Supplemental Figure S3). Additionally, deletion of FIG4 in fab1Δ VAC14-FLAG cells prevented coIP of Vac14-FLAG with the HA-tagged GroL-like fragment (B), which recapitulated the requirement for Fig4 to stabilize the interaction between Vac14 and full-length Fab1 ().
Figure 7. The GroL-like domain of Fab1 is sufficient to bind Vac14 and Fig4. (A and B) IP with anti-HA antibodies from whole cell lysates derived from fab1Δ VAC14-FLAG cells expressing empty vector, HA-GroL, or the mutated HA-GroLT/I domain (A) or from (more ...)
To further corroborate the interaction between Vac14 and the GroL-like region in vivo, we used fluorescence microscopy. To circumvent the cytosolic distribution of the GroL-like region of Fab1 (Supplemental Figure S2B), we engineered a chimeric protein composed of the GroL-like domain and of the first 134 amino acids of ALP (ALP134
contains the transmembrane domain and the AP3-pathway sorting motifs that target ALP to the vacuole (Klionsky and Emr, 1989
; Bryant et al., 1998
). Indeed, GFP-GroL-ALP was clearly targeted to the limiting membrane of the vacuole (Supplemental Figure S2B).
Expression of HA-ALP in fab1Δ cells failed to mobilize Vac14-GFP to the vacuole (, C and D). In sharp contrast, HA-GroL-ALP efficiently recruited Vac14-GFP to the vacuole. Introducing the ILLLA mutation into HA-GroL-ALP (which generated HA-GroLT/I-ALP) resulted in loss of Vac14-GFP localization to the vacuole (C). Comparing the V/C ratio for Vac14-GFP in cells expressing HA-ALP, HA-GroL-ALP, or HA-HA-GroLT/I-ALP substantiated our conclusions. The Vac14-GFP V/C ratio in cells expressing HA-ALP and the mutated HA-GroL-ALP were 1.04 ± 0.11 (n = 29) and 0.98 ± 0.12 (n = 35), respectively, and they were statistically indistinguishable (p > 0.05; D). By comparison, the V/C ratio in HA-GroL-ALP–expressing cells was 1.72 ± 0.32 (n = 30) and was considerably greater than either HA-ALP or the mutated form (p < 0.05 for both comparisons; D). In conclusion, we propose that the GroL-like region of Fab1 is an important docking site for Vac14 and Fig4, albeit it remains possible that Vac14/Fig4 may also interact with other regions on Fab1. The exact molecular events that translate into kinase activation upon Vac14-Fig4 binding will require further analysis.
In Vitro Binding between Vac14, Fig4, and a GroL-like Fragment of Fab1
To corroborate the above-mentioned observations and test for direct interaction, we used bacterially expressed recombinant proteins. GST-Vac14 and GST-GroL-like domain were purified from bacterial extracts by using glutathione-coupled agarose beads. We also expressed and purified Vac14 and Fig4 proteins tagged with an N-terminal T7 epitope and a C-terminal HIS6
epitope (E). We first tested and confirmed Vac14 interaction with itself in vitro; T7-Vac14-HIS6
was recovered with GST-Vac14 but not with GST coupled to glutathione-agarose beads (F). Similarly, T7-Fig4-HIS6
bound to GST-Vac14 but not to GST alone (F), showing that Vac14 and Fig4 interact directly. This is consistent with yeast two-hybrid analysis showing Vac14–Vac14 and Vac14–Fig4 interactions (not illustrated; Dove et al., 2002
We also tested whether recombinant GroL-like region of Fab1 was sufficient to interact directly with T7-Vac14-HIS6 and/or T7-Fig4-HIS6. We reproducibly observed enhanced recovery of T7-Vac14-HIS6 with a GST-fusion of the GroL-like region compared with GST alone (F). We could also detect an interaction between GST-GroL and T7-FIG4-HIS6, although this interaction was weak and may reflect a nonspecific affinity by the GroL-related fragment. These results suggest that recombinant GroL-like region of Fab1 can directly bind to Vac14, and perhaps Fig4, in vitro.
In conclusion, these observations support a model where Vac14 and Fig4 bind to the GroL-like region of Fab1 to form a vacuole-bound complex in the cell to regulate PtdIns(3,5)P2 levels. Our data suggest that Vac14 and Fig4 are mutually dependent for binding to Fab1 and for association with the vacuole. This can explain how Vac14 and Fig4 are concomitantly involved in synthesis and turnover of PtdIns(3,5)P2.