Fig4 is a PtdIns(3,5)P2-specific Phosphoinositide Phosphatase
Fig4 is predicted to be a phosphoinositide phosphatase not only on the basis of genetic evidence (Gary et al., 2002
), but also on the basis of sequence homology to the known phosphoinositide phosphatase sac domain (Erdman et al., 1998
; Hughes et al., 2000a
). However, attempts to demonstrate Fig4 phosphatase activity have been unsuccessful on the basis of in vivo phosphoinositide analysis of fig4
deletion mutants (Guo et al., 1999
; Gary et al., 2002
). Therefore we elected to test directly whether Fig4 is a phosphoinositide phosphatase using phosphatase assays conducted with recombinant Fig4 protein.
His-tagged full-length Fig4 was expressed in E. coli
and purified using Ni2+
-agarose affinity resin (MATERIALS AND METHODS). Phosphoinositide phosphatase activity was then assayed using fluorescent derivatives of all four phosphoinositides synthesized in yeast (PtdIns(3)P, PtdIns(4)P, PtdIns(3,5)P2
, and PtdIns(4,5)P2
). Fluorescent derivatives of phosphoinositides have been successfully used to determine substrate preferences of recombinant phosphoinositide phosphatases, including PTEN and myotubularin (Taylor and Dixon, 2001
), and myotubularin-related proteins 1 and 2 (Buj-Bello et al., 2002
; Nelis et al., 2002
). His-tagged Fig4 phosphatase activity was observed only against PtdIns(3,5)P2
and only when 1 mM magnesium was included in the assay mixture (). The reaction product of Fig4–mediated dephosphorylation migrated with fluorescent PtdIns(3)P and not PtdIns, indicating that only a single phosphate was removed from the inositol head group of PtdIns(3,5)P2
. Surprisingly PtdIns(3)P was not a substrate for Fig4 phosphatase (). We were concerned that fluorescent PtdIns(3)P might be a poor substrate for sac domain phosphoinositide phosphatases. However, His-tagged Sac1p(1–507) (Taylor and Dixon, 2001
) readily dephosphorylated fluorescent PtdIns(3)P (our unpublished results).
Figure 1. Fig4 is a magnesium-activated PtdIns(3,5)P2-specific phosphoinositide phosphatase. (A) Recombinant His-tagged full-length Fig4 was assayed in the absence and presence of 1 mM magnesium chloride using fluorescent PtdIns(3)P and PtdIns(3,5)P2 as described (more ...)
A mutant allele of FIG4, fig4-1
, was originally identified from a genetic screen as a suppressor of vac7
Δ mutant phenotypes. fig4-1 contains an arginine instead of glycine at amino acid 519, a position that is located within the seventh conserved motif of the sac domain (Gary et al., 2002
). Consequently, the mutation harbored in fig4-1 is predicted to ablate catalytic activity of the Fig4 phosphatase (Gary et al., 2002
). We next determined the in vitro phosphatase activity of His-tagged fig4-1 using PtdIns(3,5)P2
as substrate. In the presence of 1 mM magnesium, fig4-1 was unable to catalyze the dephosphorylation of PtdIns(3,5)P2
(). This result not only confirms the prediction that fig4-1 is an inactive phosphatase (Gary et al., 2002
), but also eliminates the trivial possibility that magnesium in combination with an unidentified E. coli
protein is responsible for catalyzing the dephosphorylation of PtdIns(3,5)P2
in our in vitro phosphatase assays.
Having determined the conditions required to measure Fig4 phosphatase activity in vitro, we next determined whether Fig4 dephosphorylated PtdIns(4)P and PtdIns(4,5)P2. Neither phosphoinositides were Fig4 phosphatase substrates (); therefore we conclude that Fig4 is a magnesium-activated PtdIns(3,5)P2-specific phosphatase.
Fig4 Localizes to the Vacuole Membrane
Having demonstrated that Fig4 is a PtdIns(3,5)P2
phosphatase in vitro, we next sought to understand the regulatory mechanisms that govern the cellular localization of Fig4. To begin to address this question, we wanted to observe the cellular localization of Fig4 in vivo. To achieve this, the chromosomal copy of the FIG4
locus was tagged with GFP at the 3′ end. The fusion protein was expressed as a full-length protein under the control of the native FIG4
promoter and is therefore expressed at physiological levels. Fig4-GFP localized predominantly to the vacuole membrane (). Faint Fig4-GFP fluorescence was also detected in the cytoplasm (). To visualize vacuolar membranes directly, FIG4-GFP
cells were incubated with FM4-64, a lipophilic fluorescent dye that is transported into cells via the endocytic pathway and ultimately accumulates at vacuolar membranes (Vida and Emr, 1995
). Colocalization of Fig4-GFP and FM4-64–stained vacuoles was observed (), demonstrating that Fig4-GFP localizes to the limiting membrane of the vacuole.
Figure 2. Fig4 localizes to the limiting membrane of the vacuole. Wild-type cells expressing (A) Fig4-GFP or (B) fig4Δ761–879-GFP were labeled with the fluorescent dye FM4-64 for 10 min at 26°C. The dye was washed away and the cells were (more ...)
Interestingly, we noted that there appeared to be areas of concentrated Fig4-GFP fluorescence on the vacuole membrane (highlighted by white arrow heads, ). These patches of Fig4-GFP fluorescence were not coincident with areas where vacuole membranes were closely apposed (), indicating that these patches likely define areas enriched in Fig4 phosphatase.
This result is consistent with Fig4 being a peripheral membrane-associated protein that regulates the levels of PtdIns(3,5)P2 at the vacuole membrane necessary for vacuolar size control. We did not observe GFP fluorescence within intracellular punctate structures distinct from the vacuolar membrane (), indicating that little or no Fig4 is present on other structures, such as Golgi, endosomes, or endoplasmic reticulum.
Because Fig4 lacks an obvious transmembrane domain (Erdman et al., 1998
; Gary et al., 2002
), we next sought to determine the region of Fig4 that was required for vacuole localization. Fig4 orthologues contain sequences C-terminal to the sac domain devoid of any recognizable protein motif (catalytic or otherwise); we therefore reasoned that the C-terminus of Fig4 might be responsible for targeting the phosphatase to the vacuole. To address this possibility, the chromosomal copy of the FIG4
locus was tagged with GFP, such that a truncated Fig4-GFP fusion protein was generated that was deleted for 119 amino acids at the C-terminus. Analysis of the localization of Fig4Δ761–879-GFP revealed that the fusion protein was no longer localized to the limiting membrane of the vacuole, but instead was observed exclusively within the cytoplasm of the cell (). This result indicates that the C-terminus of Fig4 is required for proper protein folding and/or contains amino-acid sequences necessary to target the phosphatase domain to the vacuole.
Vac14 Recruits Fig4 to the Vacuole
We next wanted to understand how Fig4 associates with the vacuole membrane. We reasoned that one of the known regulators of the Fab1 PtdIns(3)P 5-kinase, Vac7 or Vac14, may recruit the Fig4 phosphatase to the vacuole. Consequently, the cellular localization of Fig4-GFP in FM4-64–stained vac14Δ and vac7Δ mutants was compared with that of wild-type cells (). In vac14Δ mutants, Fig4-GFP fluorescence was observed only within the cytoplasm (). No phosphatase signal was detected at the vacuole membrane (). In contrast, in vac7Δ mutants, Fig4-GFP fluorescence was observed at the limiting membrane of the grossly enlarged vacuoles, with faint fluorescence detectable within the cytoplasm (). This pattern of fluorescence was essentially identical to that of Fig4-GFP in wild-type cells (). Moreover, more intense patches of Fig4-GFP fluorescence were again observed on the vacuole; however, this time they were more pronounced because of the grossly enlarged vacuole morphology of vac7Δ mutants (highlighted by white arrow heads, ).
Figure 3. Vac14 recruits Fig4 phosphatase to the limiting membrane of the vacuole. The vacuoles of (A) vac14Δ and (B) vac7Δ mutants expressing Fig4-GFP were visualized by FM4-64 staining as described in MATERIALS AND METHODS. The localization of (more ...)
Genetic inactivation of FIG4
levels in vac7
Δ mutants and suppresses the large vacuole phenotype exhibited by vac7
Δ mutants (Gary et al., 2002
). Consequently preservation of the large vacuole phenotype of vac7
Δ mutants in vac7
mutants (compare the FM4-64 staining of wild-type cells FIG4-GFP
in with that of vac7
mutants in ) demonstrates that the Fig4-GFP fusion protein is functional in vivo.
Because the vacuoles of vac14Δ and vac7Δ mutants are both grossly enlarged, they would be expected to share the same altered physical properties. Therefore, the fact that Fig4-GFP localizes to the grossly enlarged vacuoles of vac7Δ mutants eliminates a trivial possibility that the altered vacuole properties of vac14Δ mutants, and not the direct loss of Vac14 protein, are prohibiting the association of Fig4 with the vacuole membrane.
The failure of Fig4-GFP to localize correctly in the absence of Vac14 could possibly be due to protein instability in vac14Δ mutants, i.e., the GFP fluorescence observed in vac14Δ mutants could be generated by proteolysis of the Fig4-GFP fusion. Therefore, we determined by Western blot analysis, the steady state levels of Fig4-GFP in wild-type, vac14Δ, and vac7Δ mutants (). Inspection of Western blots obtained from total cell extracts of equal loading from wild-type, vac14Δ, and vac7Δ mutants revealed that intact Fig4-GFP was expressed at comparable levels in all strain backgrounds (). Therefore, we can conclude that the absence of Vac14 does not influence Fig4-GFP stability, but rather directly influences the capacity of Fig4 phosphatase to localize to the vacuole ().
Vac14 Localizes to the Vacuole Membrane
If Vac14 determines Fig4 localization to the vacuole, then Vac14 itself also should localize to the limiting membrane of the vacuole. To observe Vac14 localization in vivo, the chromosomal copy of the VAC14 locus was tagged in frame with GFP at the 3′ end. Vac14-GFP localized almost exclusively to the vacuole membrane, with faint fluorescence detectable in the cytoplasm (). As with Fig4-GFP, we observed patches of more intense Vac14-GFP fluorescence on the vacuole membrane (highlighted with white arrows, ) that could not be accounted for by close apposition of vacuole membranes and were not detected as bright patches with FM4-64 (). This indicates that there could exist on the vacuole membrane domains enriched for Fig4 and Vac14. Colocalization of FM4-64 and Vac14-GFP fluorescence confirmed that Vac14 localized to the limiting membrane of the vacuole (). In common with Fig4-GFP fluorescence, we did not detect Vac14-GFP fluorescence within punctate structures distinct from the vacuolar membrane, indicating that Vac14 is not localized to Golgi or endosomal membranes.
Figure 4. Vac14 localizes to the limiting membrane of the vacuole. (A) Vac14-GFP–expressing wild-type cells were labeled with the fluorescent dye FM4-64 to visualize the vacuole membranes. The localization of FM4-64 and Vac14-GFP were compared by fluorescent (more ...)
FM4-64 fluorescence also revealed that the vacuole morphology of VAC14-GFP cells were equivalent to wild-type cells (compare the FM4-64 staining of FIG4-GFP cells in with that of VAC14-GFP cells in ). This demonstrates that the Vac14-GFP fusion protein is functional. Further, Vac14-GFP is expressed as a full-length protein (), and VAC14-GFP strains synthesize wild-type quantities of PtdIns(3,5)P2 ().
To directly address the possibility that the patches of Vac14-GFP and Fig4-GFP fluorescence represent late endosomes, either fusing with, or closely opposed to the vacuole membrane, we took advantage of the phenotype exhibited by class E VPS
mutants. Class E proteins are essential for formation of MVBs (reviewed in Katzmann et al., 2002
). Deletion of any of the class E VPS
genes, such as VPS4
(Babst et al., 1997
) results in the accumulation of endosomal proteins in large aberrant endosome structures, commonly referred to as the class E compartment (Piper et al., 1995
; Rieder et al., 1996
). The class E compartment can be visualized directly by FM4-64 staining and is recognizable as an intense patch or dot of fluorescence adjacent to the vacuole (Rieder et al., 1996
). Analysis of Vac14-GFP () and Fig4-GFP () localization in vps4
Δ mutants revealed that the localization pattern of both fusion proteins was unaffected in a class E VPS
mutant. GFP fluorescence remained visible on the vacuole membrane and within intense patches on the membrane (). Further, these patches of Vac14-GFP () and Fig4-GFP () fluorescence remained distinct from the FM4-64–positive class E compartment (highlighted by white arrow heads) in vps4
Δ mutants. This result demonstrates that Vac14-GFP and Fig4-GFP are not localized to endosomes.
Figure 5. Vac14 and Fig4 do not localize to the E compartment of vps4Δ mutants. The vacuoles and the E compartments of vps4Δ mutants expressing either Vac14-GFP (A) or Fig4-GFP (B) were visualized by FM4-64 staining. The localization of the GFP (more ...)
We next determined the localization of Vac14-GFP in fig4
Δ mutants and vac7
Δ mutants. Compared with wild-type cells, Vac14-GFP fluorescence was more readily observed in the cytoplasm of fig4
Δ cells (). However, Vac14-GFP remained at the limiting membrane of the vacuole, and the distinctive patches of vacuole Vac14-GFP fluorescence remained visible in fig4
Δ mutants (). This result indicates that Vac14 localization to the vacuole membrane is not absolutely dependent on the presence of the Fig4 phosphatase. Consistent with this interpretation, fig4
Δ mutants do not exhibit phenotypes exhibited by Δvac14
mutants (Gary et al., 2002
). Therefore, Vac14 is functional in fig4
Δ mutants. However, the result does indicate that the localization of Vac14 to the limiting membrane of the vacuole is optimized by the presence of Fig4. In contrast, Vac14-GFP fluorescence was observed almost exclusively at the limiting membrane of the grossly enlarged vacuoles of vac7
Δ mutants (). Furthermore, intense patches of Vac14-GFP fluorescence were again observed on the vacuole (). This pattern of fluorescence was essentially identical to that of Vac14-GFP in wild-type cells (). This result indicates that Vac7 does not control the vacuole localization of either Vac14 or Fig4.
Vac14 localizes to the vacuole more efficiently when associated with Fig4. Vac14-GFP localization was determined in FM4-64–labeled fig4Δ (A) or vac7Δ (B) mutants by fluorescence microscopy.
Fab1 Kinase Is Required for Vacuole Localization of both Vac14 and Fig4
Bonangelino et al.
) reported that significantly reduced amounts of Vac14 fractioned with the vacuoles of fab1
Δ mutants. Consequently, we determined the localization of Vac14-GFP and Fig4-GFP in fab1
Δ mutants. Both Vac14-GFP () and Fig4-GFP () failed to localize to the grossly enlarged vacuoles of fab1
Δ mutants. Taken together, our results indicate that Fab1 kinase, but not Vac7, regulates the vacuole localization of Vac14 and Fig4. However, because we and Bonangelino et al.
) have been unsuccessful in detecting a direct protein-protein interaction between Fab1 and Vac14, additional proteins are most likely required to mediate Vac14 localization to the vacuole.
Fab1 kinase is required for vacuole localization of Vac14 and Fig4. (A) Vac14-GFP and (B) Fig4-GFP localization were determined in FM4-64–labeled fab1Δ mutants by fluorescence microscopy.
Fig4 Interacts with Vac14 at the Vacuole Membrane
To date, no physical interactions have been detected between any one of the proteins that constitute the known machinery for regulation of PtdIns(3,5)P2 levels, namely the Fab1 kinase, Vac7, Vac14, and Fig4 phosphatase. However, the Vac14-dependent association of Fig4 with vacuole membranes () and the vacuolar localization of Vac14 () indicates that Vac14 may physically interact with Fig4. To test this directly, we determined whether both proteins could be coimmunoprecipitated in a common protein complex. Lysates from wild-type Vac14-GFP–expressing cells were fractionated into P13, P100, and S100 fractions. Vac14-GFP was immunoprecipitated from the detergent-solubilized P13 and P100 fractions, but not from the S100 fraction (). Western blot analysis was then undertaken to determine whether Fig4 coimmunoprecipitated with Vac14-GFP. Fig4 was detected in immunoprecipitates of Vac14-GFP from both the P13 and P100 fractions (). Fig4 did not fractionate to the S100 fraction (). These results suggest that Vac14 and Fig4 physically associate within a common membrane associated protein complex.
Figure 8. Vac14 interacts with Fig4. Coimmunoprecipitation of Vac14-GFP and Fig4 from P13 and P100 fractions. Lysates from wild-type cells expressing Vac14-GFP were fractionated into P13, S100, and P100 fractions, and Vac14-GFP immunoprecipitated from detergent-solubilized (more ...)
Fig4 Mislocalization in vac14Δ Mutants Does Not Contribute to vac14Δ Phenotypes
Δ mutants are unable to synthesize wild-type levels of PtdIns(3,5)P2
(Bonangelino et al., 2002
; Dove et al., 2002
) and Fab1 kinase is localized to both endosomes and vacuole membranes (Gary et al., 1998
; Bonangelino et al., 2002
; Dove et al., 2002
), we wanted to ascertain whether or not the mislocalization of the PtdIns(3,5)P2
-specific Fig4 phosphatase contributed to the phenotypes exhibited by vac14
Δ mutants. We reasoned that an increased level of nonvacuolar Fig4 could in theory gain access to endosomal pools of PtdIns(3,5)P2
. Therefore, we deleted FIG4
Δ strains and examined both the vacuole morphology and phosphoinositide levels of vac14
Δ double mutants. FM4-64 staining of the double mutants revealed that vac14
Δ double mutants exhibited single grossly enlarged vacuoles (). In vivo phosphoinositide analysis of vac14
Δ double mutants revealed that PtdIns(3,5)P2
levels were identical to those of vac14
Δ single mutants (). Therefore, this result indicates that mislocalized Fig4 does not contribute to the phenotype exhibited by vac14
Mislocalized Fig4 phosphatase does not contribute to vac14Δ phenotypes. Vacuoles of wild-type, fig4Δ, vac14Δ, and vac14Δ fig4Δ strains were visualized by FM4-64 staining and fluorescence microscopy.
3′-Phosphoinositide levels in wild-type, fig4Δ, vac14Δ, and vac14Δ fig4Δ mutants
Genetic inactivation of FIG4
suppresses the phenotypes associated with vac7
Δ mutants (Gary et al., 2002
); however, vac14
Δ double mutants exhibit identical phenotypes to that of vac14
Δ mutants (). Consistent with this result, there was no increased PtdIns(3,5)P2
synthesis detected in vac14
Δ double mutants compared with vac14
Δ mutants (). Therefore, fig4
Δ mutations neither contribute to, nor suppress vac14
The failure of fig4Δ to suppress vac14Δ might be accounted for by the continued turnover of PtdIns(3,5)P2 by Sjl2, Sjl3, and/or SacI. However, deletion of these sac domain-containing proteins also failed to suppress vac14Δ (our unpublished results). Taken together, these results indicate that in the absence of Vac14, Vac7-dependent activation of Fab1 kinase is attenuated. Therefore, Vac14 is required for maximal Vac7-dependent activation of the Fab1 kinase.
Vac14 Is Required for fig4Δ Suppression of vac7Δ
The data presented demonstrate that Vac14 functions to recruit Fig4 to the vacuole membrane. However, if this were the sole role of Vac14, then one would predict that vac14
Δ mutants would synthesize increased amounts of PtdIns(3,5)P2
. Instead vac14
Δ mutants are unable to synthesize wild-type levels of PtdIns(3,5)P2
(Bonangelino et al., 2002
; Dove et al., 2002
) and vac7
Δ double mutants exhibit the same phenotypes as both the single vac7
Δ and vac14
Δ deletion mutants (Bonangelino et al., 1997
). Moreover, from our analysis of vac14
Δ double mutants in this article, we have data that indicate that Vac14 is required for maximal Vac7-dependent activation of the Fab1-kinase. Taken together, these results indicate that Vac14 also performs a second cellular function of positively regulating Fab1 kinase activity, in addition to recruiting Fig4 phosphatase to the vacuole.
To test whether Vac14 regulates Fab1 kinase activity, we reasoned that deletion of VAC14 from vac7Δ fig4Δ double mutants would eliminate Fab1 kinase activity. Analysis of vac7Δ fig4Δ vac14Δ triple mutants revealed that these mutants exhibited vac7Δ phenotypes; enlarged vacuoles () and in vivo phosphoinositide analysis determined that these mutants synthesized undetectable levels of PtdIns(3,5)P2 (). Consequently, Vac14 is required for PtdIns(3,5)P2 synthesis in the absence of Vac7 and Fig4. Therefore, Vac14 positively regulates Fab1 kinase. This additional function of Vac14 explains why vac14Δ mutants fail to suppress vac7Δ mutants.
Figure 10. Vac14 is required for fig4Δ suppression of vac7Δ. The vacuole morphologies of wild-type, vac7Δ, vac7Δ fig4Δ, and vac7Δ fig4Δ vac14Δ strains were visualized by fluorescent staining with FM4-64 (more ...)
3-Phosphoinositide levels in wild-type, vac7Δ, vac7Δ fig4Δ, and vac7Δ fig4Δ vac14Δ mutants