PIKfyve-ArPIKfyve-Sac3 Core Complex

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P2 biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P2 hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyveK1831E if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P2. In contrast, PIKfyveK1831E, which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyveWT-Sac3WT but minimal defects when coexpressed with ArPIKfyveWT and phosphatase-deficient Sac3D488A. These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P2 phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.

The seven phosphorylated derivatives of phosphatidylinositol (PtdIns), 2 called collectively PIs, are eukaryotic membrane-anchored signaling molecules that orchestrate diverse cellular processes, including intracellular membrane trafficking (1)(2)(3)(4)(5)(6). PtdIns(3,5)P 2 , a low abundance PI comprising as little as 0.8% of total PIs in mammalian cells, mediates essential aspects of endocytic membrane homeostasis (7). Although mechanistic details remain to be elucidated, experimental evidence indicates that PtdIns(3,5)P 2 may coordinate fission and fusion events in the multivesicular endosomal system of mammalian cells (8,9). Consistent with these roles, perturbations in PtdIns(3,5)P 2 production impair several intracellular trafficking pathways, both constitutive and regulated, that emanate from or traverse the early endosomes (10 -13). In line with the requirement for PtdIns(3,5)P 2 in maintaining proper balance between membrane removal (fission) and membrane insertion (fusion), disrupted function of PIKfyve, the sole enzyme for PtdIns(3,5)P 2 synthesis, is phenotypically manifested by endosome vesicle swelling and endomembrane vacuolation seen in a number of mammalian cell types (7). As unraveled recently, PIKfyve is engaged in an unusual physical interaction with the phosphatase Sac3 that turns over PtdIns(3,5)P 2 , forming a common endogenous complex (the PAS core complex) organized by the PIKfyve activator ArPIKfyve (9,14). The ternary association, scaffolded by ArPIKfyve homomeric interactions, activates the PIKfyve kinase as evidenced by recent data for reduced PIKfyve activity upon disintegration of the PAS core (14). However, whereas the assembly of the three proteins in the PAS core is critical for PIKfyve activation and regulated PtdIns(3,5)P 2 production, whether the same complex is a functional platform for Sac3 enzymatic activity is currently unknown.
PIKfyve, ArPIKfyve, and Sac3 are large, evolutionarily conserved proteins encoded by single-copy genes from yeast to humans. They all incorporate a range of functional domains (7). In the case of PIKfyve, there is an N-terminal-positioned FYVE finger domain that targets the protein to PtdIns(3)Penriched endosome membranes (15). Next is the DEP domain, still with an uncharacterized function. The middle part of the molecule (residues 560 -1438) is occupied by two domains: Cpn60_TCP1, with sequence similarity to molecular chaperonins, and a CHK homology region, with conserved Cys, His, and Lys residues uniquely displayed by the PIKfyve orthologs (7). The region of conserved Lys is homologous to spectrin repeats. At the C terminus is the catalytic domain, responsible for the three PIKfyve kinase activities, i.e. synthesis of PtdIns(3,5)P 2 , PtdIns(5)P, and phosphoproteins, including phospho-PIKfyve (16,17). The aberrant endomembrane vacuolar phenotype has been first observed upon ectopic expression of kinase-deficient PIKfyve with a point mutation in the predicted ATP binding Lys 1831 of the catalytic domain (18). Similar defects have been confirmed thereafter by small interfering RNA-mediated silencing and pharmacological inhibition of PIKfyve in different mammalian cell types (13,19). All these maneuvers, however, although preserving the proper intracellular localization of the enzyme (15), affect all three PIKfyve kinase activities. That the aberrant vacuolar phenotype is due to abrogated PtdIns(3,5)P 2 synthesis is evidenced by its appearance in cells expressing PtdIns(3,5)P 2 -deficient, but not PtdIns(5)P-deficient, PIKfyve point mutants and its subsequent reversal upon exogenous delivery of PtdIns(3,5)P 2 (17). Importantly, aberrant endomembrane vacuoles are no longer seen if PtdIns(3,5)P 2 -deficient PIKfyve K1831E is mislocalized by disruption of its FYVE finger (18). Clearly, these data corroborate the conclusion that if properly localized PIKfyve mutants fail to produce PtdIns(3,5)P 2 , they can then trigger dominant-negative changes in mammalian cells, phenotypically manifested by endomembrane vacuoles. One puzzling observation awaiting clarification is the apparent inability of a Cpn60_TCP1-deletion PIKfyve mutant to induce endomembrane vacuoles despite its proper intracellular localization and lack of in vitro lipid kinase activity (18). Considering the partner Sac3 phosphatase collaborating in parallel with PIKfyve in triggering the endomembrane defects by reducing localized PtdIns(3,5)P 2 levels, here we examined the interacting regions of the three proteins and their functional significance to the endomembrane homeostatic mechanism. We report here that the Cpn60_TCP1 domain of PIKfyve is a major determinant in recruiting the ArPIKfyve-Sac3 subcomplex. A PIKfyve mutant truncated in this domain is not only incapable of binding the ArPIKfyve-Sac3 subcomplex but also unable to induce aberrant endomembrane vacuoles even if harboring the kinase-dead K1831E mutation. Concordantly, the PIKfyve K1831E point mutant whose binding to ArPIKfyve-Sac3 is intact largely loses its ability to vacuolate cells when coexpressed with phosphatase-deficient Sac3 D488A and ArPIKfyve but exacerbates the defective vacuolar phenotype when coexpressed with active Sac3 WT and ArPIKfyve. These data indicate that Sac3 relays hydrolyzing activity from the PAS core, greatly contributing to the endomembrane defects associated with the PIKfyve inactivation and PtdIns(3,5)P 2 deficiency.
Cell Cultures and Transfections-COS7 cells were cultured and transiently transfected with the indicated cDNAs by Lipofectamine 2000 or Lipofectamine (Invitrogen) for immunoprecipitation and immunofluorescence microscopy analyses, respectively, under conditions described in previous studies (14,18).
Immunofluorescence and Light Microscopy-Transfected COS7 cells grown on coverslips were subjected to immunofluorescence microscopy analysis 12 and/or 24 h post-transfection as described previously (8,18). Permeabilized cells were stained with the primary and secondary antibodies indicated in the figure legends. Coverslips were mounted on slides using the Slow Fade Antifade kit (Invitrogen). Cells were viewed in a Nikon Eclipse TE200 inverted microscope equipped with a Plan Apo 60 ϫ 1.4 Ph3DM oil objective and 3 standard fluorescence channels (i.e. green for GFP, red for anti-Myc/Alexa568, and blue for anti-Sac3/Alexa350 signals). Images were captured with a SPOT RT slider charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI) and processed using SPOT 3.2 and Adobe Photoshop 6.0 software. Colocalization with EEA1 was investigated by confocal microscopy (model 1X81, Olympus) by a 60ϫ UplanApo lens. Images were captured by a cooled charge-coupled device 12-bit camera (Hamamatsu) as described previously (8,9). Baculoviral Vectors, Recombinant Protein Expression/Purification, and in Vitro Binding-Generations of GST-His 6 -mPIKfyve in baculoviral expression vector pAcGHLT-A and that of His 6 -hArPIKfyve in bacterial expression vector pRSETb were described previously (16,21). The baculoviral expression vector pFASTBac-GST-hSac3 was a kind gift by Dr. Takenawa. His 6 -ArPIKfyve and Sac3 were simultaneously expressed using a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression (23) as detailed in supplemental Material 3. For the in vitro binding assays, GST-PIKfyve or His 6 -PIKfyve (ϳ15 ng protein) purified and immobilized on GSH-or Ni-NTA-agarose as described elsewhere (16) was incubated for 2-16 h with His 6 -ArPIKfyve or GST-Sac3, respectively, produced and purified separately. Immobilized GST-PIKfyve was incubated for 2 h with the His 6 -ArPIKfyve-Sac3 subcomplex, purified from infected Sf21 cells on a Ni-NTA column. Beads were washed following previously published protocols (24). The reactions were analyzed by SDS-PAGE and immunoblotting.
Immunoprecipitation and Immunoblotting-Fresh cell lysates, collected in RIPA ϩ buffer (50 mM Tris/HCl buffer, pH 8.0, containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 1ϫ protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml aprotinin, 1 g/ml pepstatin, and 1 mM benzamidine)) were precleared (20,000 ϫ g, 15 min, 4°C) and subjected to immunoprecipitation 24 h post-transfection. Control immunoprecipitates with nonimmune rabbit or mouse IgG were run in parallel. Immunoprecipitations were carried out for 16 h at 4°C with protein A-Sepharose CL-4B added in the final 1.5 h of incubation. Immunoprecipitates were washed five times with RIPA ϩ buffer and then processed by Western blotting. Immunoblotting with the antibodies indicated in the figure legends was performed subsequent to protein separation by SDS-PAGE (typically on 6% gels) and electrotransfer onto nitrocellulose membranes as described previously (14,21). A chemiluminescence kit (Pierce) was used to detect the horseradish peroxidase-bound secondary antibodies.
Lipid and Protein Kinase Activity in Vitro Assays-PIKfyve lipid and protein kinase activities were measured in parallel using PIKfyve immunoprecipitates derived from transfected cells. For the lipid kinase assay, washed PIKfyve immunoprecipitates were subjected to 15 min of incubation at 37°C in an assay buffer (50 mM Tris, pH 7.4, 2.5 mM MgCl 2 , and 2.5 mM MnCl 2 ) supplemented with 50 M ATP, [␥-32 P]ATP (12.5 Ci), and 100 M PtdIns (from soybean, Avanti Polar Lipids Inc) as detailed elsewhere (14,22). Lipids were extracted and analyzed by silica gel TLC using an acidic solvent system (2 M acetic acid/propanol, 1:2 v/v). Both PtdIns(5)P-and PtdIns(3,5)P 2synthesized products were monitored simultaneously as we detailed elsewhere (21,22). PIKfyve protein kinase was measured by the PIKfyve autophosphorylation activity in a reaction mix composed of [␥-32 P]ATP (5 Ci), 25 M ATP, 24 mM MgCl 2 , and 5 mM MnCl 2 in 50 mM Hepes, pH 7.4, and carried out for 30 min at 25°C as described elsewhere (12,16). Generated lipid or protein products were detected by autoradiography and quantified by radioactive counting of the scraped silica spots or cut radioactive band.
Other Methods-Protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Protein levels were quantified from the intensity of the immunoblot bands with a laser scanner (Microteck) and UN-SCAN-IT software (Silk Scientific). Several films of different exposure times were quantified to assure the signals were within the linear range. Data are expressed as mean Ϯ S.E. Statistical analysis was performed by either one-or two-tailed Student's t test.

RESULTS
ArPIKfyve and Sac3 Associate with Each Other via Their C Termini Independently of the Sac3 Phosphatase Activity-As established previously, PIKfyve interacts efficiently with ArPIKfyve and Sac3 only when the two proteins are together, whereas ArPIKfyve and Sac3 form an easily detectable heteromeric subcomplex without PIKfyve (14). That ArPIKfyve and Sac3 interact directly with each other in a manner independent of PIKfyve or other proteins was also confirmed herein in Sf21 insect cells infected with baculoviruses expressing His 6 -ArPIKfyve and Sac3 simultaneously. Purification of His 6 -ArPIKfyve on Ni-NTA-agarose resin reproducibly copurified Sac3 (supplemental Fig. 1). Therefore, to begin characterizing the contact sites in the PAS core complex, we first examined the molecular interaction of the ArPIKfyve-Sac3 subcomplex. We generated two overlapping GFP-based constructs of the Sac3 N-and C-terminal halves (Fig. 1A) and examined their efficiency to interact with Myc-ArPIKfyve WT by coimmunoprecipitation analyses in double-transfected COS cells. As illustrated in Fig. 1B, whereas the N-terminal half of Sac3 (residues 1-574) coimmunoprecipitated with Myc-ArPIKfyve WT only weakly, the C-terminal Sac3 fragment (residues 388 -907) displayed the wild-type binding efficiency. With a goal to determine the minimal region that supports the interaction with ArPIKfyve, we next assessed shorter C-terminal fragments of Sac3 (Fig. 1, A and B). Sac3-(478 -907) associated with an efficiency similar to that of Sac3-(388 -907) or Sac3 WT , whereas the Sac3-(610 -907) peptide fragment displayed 85 Ϯ 8% of the maximal binding (Fig. 1, A and B). Sac3 D488A that harbors a point mutation in the phosphatase domain to abrogate the phosphatase activity (9) displayed the Sac3 WT association efficiency ( Fig. 1, A and B). These data indicate that Sac3 interacts with ArPIKfyve WT independently of its phosphatase activity, and a C-terminal fragment of ϳ430 amino acids is required to mimic the efficiency of the Sac3 WT association with ArPIKfyve.
To determine the ArPIKfyve region interacting with Sac3, we examined two nearly overlapping GFP-based constructs of the ArPIKfyve N-and C-terminal region for their interaction with Myc-Sac3 WT by coimmunoprecipitation in double-transfected COS cells (Fig. 1C). Notably, only the fragment of the ArPIKfyve C-terminal region spanning residues 523-782 was recovered with Myc-Sac3 WT (Fig. 1D). The association of the N-terminal region was Ͻ10% of that seen with ArPIKfyve WT binding to Sac3 WT (Fig. 1D). These data indicate that the C-terminal region of ArPIKfyve spanning residues 523-782 interacts with Sac3.
Sac3 Interacts with an ArPIKfyve Homodimer-As revealed recently, ArPIKfyve forms a homodimer (or a higher-order homooligomer) that scaffolds the PAS core (14). Intriguingly, the same C-terminal fragment that interacts with Sac3 is engaged in the ArPIKfyve homomerization (14). To better understand how the ArPIKfyve-Sac3 subcomplex is organized, were analyzed by immunoprecipitation (IP) with anti-Myc or anti-GFP antibodies as indicated. C, ArPIKfyve deletion mutants are presented schematically relative to the ArPIKfyve domain architecture predicted by Pfam databases (E-value cutoff ϭ 1) and COILS. Shown is quantitation of their interaction with Sac3 WT without or with differently tagged ArPIKfyve WT and measured by co-IP as shown in D and E. D, cells cotransfected with cDNAs of Myc-Sac3 WT plus either eGFP-HA-ArPIKfyve WT , eGFP-HA-ArPIKfyve Nt , or eGFP-ArPIKfyve Ct were analyzed by IP with anti-Myc antibody are shown. E, cells double-transfected with cDNAs of Myc-ArPIKfyve WT plus eGFP-HA-ArPIKfyve WT (a control for ArPIKfyve homodimerization) or triple-transfected with cDNAs of Myc-ArPIKfyve WT , Myc-Sac3 WT , and either eGFP-HA-ArPIKfyve WT or eGFP-HA-ArPIKfyve Nt , were analyzed by IP with anti-HA antibodies. Seen is efficient co-IP of Myc-Sac3 WT with the Myc-ArPIKfyve WT -eGFP-HA-ArPIKfyve WT homodimers. B, D, and E, fresh RIPA ϩ lysates were subjected to IP, and after washes in the same buffer, IPs were analyzed by SDS-PAGE and immunoblotting with anti-GFP and anti-Myc antibodies with a stripping step in between. Shown are chemiluminescence detections of a representative experiment for each of B, D, and E panels from three to six independent determinations with S.E. Ͻ10% of the mean value, quantitatively scored in A and C. Levels of the co-IP proteins were quantified relative to their total expression amounts and then normalized for the immunoprecipitated levels relative to the wild-type, which was scored as ϩϩϩϩ. Mutants scored as Ϫ displayed Ͻ10% of the wild-type association.
we examined if ArPIKfyve could concurrently interact with Sac3 and with itself. To test this we expressed simultaneously two distinctly tagged versions of ArPIKfyve WT together with Myc-Sac3 WT . We used Myc-ArPIKfyve WT and GFP-HA-ArPIKfyve WT because they exhibit markedly different electrophoretic mobilities, making their unambiguous detections possible. Importantly, both Myc-Sac3 WT and Myc-ArPIKfyve WT were recovered in the HA-ArPIKfyve WT immunoprecipitates (Fig. 1E). The ArPIKfyve Nt fragment neither homodimerized nor associated with Sac3 WT to a significant extent, as evidenced by the lack of Sac3 WT or ArPIKfyve Nt immunoreactive bands coimmunoprecipitated with anti-HA ArPIKfyve Nt (Fig. 1E). Together, these data indicate that ArPIKfyve-ArPIKfyve and ArPIKfyve-Sac3 interactions proceed simultaneously through the same region of ArPIKfyve spanning residues 523-782. Intriguingly, this region, identified by data base searches as "Domain with unknown function," incorporates four of the predicted five coiled-coil motifs in ArPIKfyve (Fig. 1C), conducive in forming coiled-coil helical backbone for structural stability of multiprotein interactions (25). Because Sac3 does not form homooligomeric structures (14), seen also with the yeast counterpart (26), these data are consistent with the notion that the subcomplex incorporates ArPIKfyve and Sac3 at a molar ratio of 2 (or higher) to 1.
ArPIKfyve and Sac3 N Termini Interact with PIKfyve-We next sought to determine the regions in ArPIKfyve-Sac3 interacting with PIKfyve. As found previously in ectopically transfected COS cells, PIKfyve efficiently interacts with ArPIKfyve and Sac3 only when the two are together (14). This conclusion was corroborated herein by the data from in vitro reconstituted binding of the three recombinant proteins, expressed and purified from insect or bacterial cells. Thus, GST-PIKfyve immobilized on GSH-agarose did not pull down His 6 -ArPIKfyve to a significant degree ( Fig. 2A). Likewise, His 6 -PIKfyve immobilized on Ni-NTA-agarose did not pull down GST-Sac3 (not shown). However, if the His 6 -ArPIKfyve-Sac3 subcomplex, purified from infected Sf21 on Ni-NTA-agarose, was incubated with purified GST-PIKfyve immobilized on GSH beads, both Sac3. GST-PIKfyve or GST control purified from infected Sf9 cells and immobilized on GSH-agarose beads was incubated with bacterially produced His 6 -ArPIKfyve or His 6 -GDI2 (as a control), both purified on Ni-NTA-agarose (left panels), or with His 6 -ArPIKfyve-Sac3 subcomplex, purified on Ni-NTA-agarose from infected Sf21 cells (right panels) as indicated. Blots, cut in the middle, were probed with anti-PIKfyve (upper halves) or anti-ArPIKfyve and anti-Sac3 antibodies (lower halves) with a stripping step between the latter two. Shown are representative blots illustrating that efficient associations occur only with the three purified recombinant proteins. The asterisk (*) depicts incompletely stripped His 6 -ArPIKfyve. B, COS7 cells were triple-transfected with cDNAs encoding Myc-ArPIKfyve WT , HA-PIKfyve WT , and either eGFP-Sac3 WT , eGFP-Sac3-(388 -907), or eGFP-Sac3-(610 -907). Fresh RIPA ϩ lysates underwent IP with anti-Myc antibody, and after washes in the same buffer, IPs were analyzed by SDS-PAGE and immunoblotting with anti-GFP, anti-Myc, and anti-HA-antibodies with a stripping step in between. Shown are chemiluminescence detections of a representative transfection experiment from three independent determinations with S.E. Ͻ10% of the mean. C, shown is a schematic diagram of the PIKfyve domain structure, predicted by Pfam (E-value cutoff ϭ 1) or BLAST databases and a quantitative summary of the PIKfyve interaction with the complexes of ArPIKfyve WT -Sac3 WT , ArPIKfyve WT -Sac3-(610 -907), ArPIKfyve WT -Sac3(388 -907), ArPIKfyve-(523-782)-Sac3 WT , and ArPIKfyve-(298 -782)-Sac3 WT .
His 6 -ArPIKfyve and Sac3 were readily pulled down ( Fig. 2A). Therefore, to determine how PIKfyve associates with the ArPIKfyve-Sac3 subcomplex, we triple-transfected COS cells with constructs of appropriately tagged PIKfyve WT , ArPIKfyve WT , and C-terminal fragments of Sac3 or PIKfyve WT , Sac3 WT , and C-terminal fragments of ArPIKfyve. Coimmunoprecipitation was carried out with antibodies against the tags in ArPIKfyve WT or Sac3 WT . Levels of retrieved PIKfyve WT were quantified relatively to those found with the ArPIKfyve-Sac3 wild type. As illustrated in Fig. 2B, Sac3-(610 -907) and ArPIKfyve WT poorly associated with PIKfyve WT as judged by the low HA-PIKfyve WT amounts coimmunoprecipitated with anti-Myc-ArPIKfyve WT . Likewise, a longer Sac3 fragment spanning residues 388 -907 did not bring about greater levels of HA-PIKfyve WT retrieved in Myc-ArPIKfyve WT immunoprecipitates (Fig. 2, B and C). As expected, however, complexes between ArPIKfyve WT and the Sac3 C-terminal fragments were readily formed at the efficiency similar to that seen with the Sac3 wild type (Fig. 2, B and C). This observation indicates that even though a complex between ArPIKfyve and C-terminal Sac3 is formed, PIKfyve is not efficiently retrieved, consistent with the requirement of the Sac3 N-terminal region (1-388) in the PAS core formation. Likewise, ArPIKfyve-Sac3 complexes made of Myc-Sac3 WT and GFP-ArPIKfyve-(523-782) or GFP-ArPIKfyve-(288 -782) did not efficiently retrieve HA-PIKfyve WT (quantified in Fig. 2C), indicating the ArPIKfyve N-terminal 1-288 residues are required for an efficient PIKfyve incorporation within the PAS complex. These observations further indicate that the PAS complex could not be formed in the absence of ArPIKfyve or Sac3 N termini even though a stable subcomplex via their C termini can still be formed.
PIKfyve Cpn60_TCP1 and CHK Homology Are Essential in Interacting with the ArPIKfyve-Sac3 Subcomplex-To examine how PIKfyve associates with the ArPIKfyve-Sac3 subcomplex in the PAS core, we generated several GFP-HA or HA-based truncated mutants of PIKfyve (Fig. 3A). Their efficiency to interact with the wild-type ArPIKfyve-Sac3 subcomplex was examined relative to that of PIKfyve WT by coimmunoprecipitation in triple-transfected COS cells. We began characterizing the binding efficiency of a truncated mutant with deleted Cpn60_TCP1 domain and N-terminal parts of the CHK homology (deleted residues 560 -1231; Fig. 3A). The CHK homology (residues 1151-1461), shared by all PIKfyve orthologs, harbors conserved Cys (6 versus Fab1) or His (4 versus Fab1), positioned at the N-terminal half (residues 1155-1327), and Lys (11 versus Fab1), positioned at the C-terminal half of the homology (1295-1461). Sequence analyses by modular databases identify the region of conserved Lys as spectrin repeats (residues 1367-1464; Fig. 3A), a module that supports assembly in multiprotein complexes involved in cytoskeletal architecture and signal transduction (27). Therefore, we herein refer to the N-terminal and C-terminal part of the CHK homology as CH and spectrin repeats, respectively (Fig. 3A). Intriguingly, for reasons awaiting clarification, the ⌬560 -1231 mutant (herein as ⌬Cpnϩ) was known for quite some time to be devoid of lipid kinase activity (22) despite the intact lipid kinase domain (Fig. 3A). This peculiar observation points to the Cpn60_TCP1 and/or CH homology domains as a plausible candidate region for binding the ArPIKfyve-Sac3 subcomplex, thereby activating PIKfyve kinase activity. This suggestion was corroborated herein by coimmunoprecipitation analyses, shown in Fig.  3B, in which we detected only insignificant amounts of Myc-ArPIKfyve WT and Myc-Sac3 WT (Fig. 3A) in HA-PIKfyve ⌬Cpnϩ immunoprecipitates. Mutants with shorter truncations in the Cpn60_TCP1 domain, namely ⌬560 -749 (herein as ⌬N-Cpn) and ⌬807-1032 (herein as ⌬C-Cpn), also showed a markedly reduced ability of retrieving Myc-ArPIKfyve WT and Myc-Sac3 WT (Fig. 3, A and C), indicative for the presence of binding determinants on both the Nand C-terminal halves of the Cpn60_TCP1 domain. In contrast, the FYVE-finger deletion mutant interacted with ArPIKfyve-Sac3 at the wild-type efficiency, as judged by the similar levels of Myc-ArPIKfyve WT and Myc-Sac3 WT immunoreactive bands recovered in immunoprecipitates of HA-PIKfyve ⌬FYVE versus HA-PIKfyve WT (Fig. 3, A and B).
To confirm the critical role of the Cpn60_TCP1/CH homology domains in binding the ArPIKfyve-Sac3 subcomplex, we sought to reconstitute the triple association in ectopically transfected COS cells. We assessed the efficiency at which ArPIKfyve WT and Sac3 WT coimmunoprecipitated with a PIKfyve fragment spanning residues 1-1260. This mutant, referred to herein as PIKfyve ⌬Spec⌬Kinϩ , harbors the region of Cpn60_TCP1 and CH deleted in the ⌬Cpnϩ mutant, but the C-terminal part downstream of the CH homology encompassing the spectrin repeats and the catalytic domain is eliminated (Fig. 3A). Surprisingly, the PIKfyve ⌬Spec⌬Kinϩ mutant was incapable of reproducing the efficiency of the PAS wild-type associations as evidenced by only 25% potency of PIKfyve ⌬Spec⌬Kinϩ versus PIKfyve WT immunoprecipitates to retrieve ArPIKfyve WT and Sac3 WT (Fig. 3, A and D). Likewise, a larger fragment that incorporates the entire CH/spectrin repeats homology along with the Cpn60_TCP1 domain, referred to herein as ⌬F⌬Kinϩ, was unable to coimmunoprecipitate Myc-ArPIKfyve WT and Myc-Sac3 WT to an extent seen with PIKfyve WT (Fig. 3, A and E). These data indicate that the region encompassing Cpn60_TCP1 and upstream of CH is a necessary binding determinant, yet alone it is insufficient to associate with the ArPIKfyve-Sac3 subcomplex as efficiently as PIKfyve wild type.
The PIKfyve Kinase Domain, but Not Activity, Is Required for Interaction with the ArPIKfyve-Sac3 Subcomplex-The observation that a peptide fragment of the N-terminal and middle region of PIKfyve incorporating the Cpn60_TCP1 domain and upstream of the CH homology is insufficient to mimic the PIKfyve WT association with ArPIKfyve-Sac3 suggests the C-terminal region downstream of the CH homology also plays a role in the PAS core formation and stabilization. To test this we examined the interaction of a truncated mutant harboring nearly the entire PIKfyve sequence minus the C-terminal kinase domain (Fig. 3A). As illustrated in Fig. 3, A and F (Fig. 3, A and F). Consistent with the requirement for the catalytic domain in productive ternary complex formation, a PIKfyve C-terminal fragment spanning residues 930 -2052 (referred to as ⌬F⌬D⌬Cpn), interacted to some extent with ArPIKfyve-Sac3 as judged by the detection of Myc-ArPIKfyve WT and Myc-Sac3 WT in HA-PIKfyve ⌬F⌬D⌬Cpn immunoprecipitates (Fig. 3, A  and G). It should be emphasized, however, that either of the two overlapping PIKfyve halves (residues 1-1260 and 930 -2052) produced only ϳ 1 ⁄ 4 of the PIKfyve wild-type association with ArPIKfyve-Sac3 (Fig. 3A), suggesting ArPIKfyve/Sac3-induced alterations in the PIKfyve conformation to optimally accommodate the ArPIKfyve-Sac3 subcomplex. Together, these data are consistent with the notion that ArPIKfyve-Sac3 first docks at the Cpn60_TCP1/CH region, which induces conformational changes in PIKfyve to uncover additional binding sites stabilizing the ternary associations of the PAS core.
No Phenotypic Defects by PIKfyve K1831E If Its ArPIKfyve-Sac3 Binding Region Is Eliminated-Having identified the Cpn60_TCP1/CH region as a major determinant in associating with ArPIKfyve-Sac3, we next assessed the outcome of the lost ArPIKfyve-Sac3 binding to the genuine PIKfyve characteristics, such as lipid or protein kinase activities, intracellular localization, and the effect on endomembrane homeostasis. Intriguingly, the PIKfyve ⌬Cpnϩ mutant was devoid of measurable in vitro lipid kinase activity as observed previously (22) and confirmed herein under longer exposure times (4 days) of the autoradiograms (Fig. 4A, quantified in Fig. 3A). However, PIKfyve ⌬Cpnϩ displayed in vitro protein kinase activity comprising 20 -25% that determined for PIKfyve WT (Fig. 4A), unlike the PIKfyve K1831E mutant that lacked any lipid or protein kinase activities (16). These data suggest that associated ArPIKfyve-Sac3 is essential for PtdIns(3,5)P 2 and PtdIns(5)P synthesis but less critical for the protein kinase activity of PIKfyve. This conclusion is also substantiated by data demonstrating that if PAS-associated levels of endogenous ArPIKfyve are reduced by small interfering RNA-mediated knockdown or detergent stripping, the PIKfyve lipid kinase activity is markedly inhibited, whereas the autokinase activity remains unaltered (12, 21, and data not shown).
We also examined the lipid kinase activity of other PIKfyvetruncated mutants (supplemental Fig. 2) that displayed intact kinase domain but reduced ArPIKfyve-Sac3 binding and inspected the ability of the mutant to induce vacuolar defects. Quantitative summary of these observations from at least three independent experiments for each construct is presented in Fig.  3A. The comparative analysis allows two important conclu-  COS7 cells (shown in B-G), the in vitro lipid kinase activity determined herein (see Fig. 4A and supplemental Fig. 2) or in the indicated references, and their ability to induce vacuolar defects determined herein (see Fig.  4B and data not shown) or in the indicated references. CHK homology (residues 1155-1461) harbors conserved Cys and His within its N-terminal half (residues 1155-1327) and conserved Lys within its C-terminal half (residues 1295-1461) that is homologous to spectrin repeats. B-G, COS7 cells were triple-transfected with Myc-Sac3 WT , Myc-ArPIKfyve WT , and the following PIKfyve constructs: HA-PIKfyve WT , HA-PIKfyve ⌬Cpnϩ , or eGFP-HA-PIKfyve ⌬FYVE (B); eGFP-HA-PIKfyve WT , eGFP-HA-PIKfyve ⌬C-Cpn , or eGFP-HA-PIKfyve ⌬N-Cpn (C); HA-PIKfyve WT or HA-PIKfyve ⌬Spec⌬Kinϩ (D); eGFP-HA-PIKfyve WT or eGFP-HA-PIKfyve ⌬F⌬Kinϩ (E); HA-PIKfyve WT , HA-PIKfyve K1831E , or HA-PIKfyve ⌬Kin (F); eGFP-HA-PIKfyve WT or eGFP-HA-PIKfyve ⌬F⌬D⌬Cpn (G). Anti-HA IPs from fresh RIPA ϩ lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA, anti-Myc, and anti-GFP antibodies, with a stripping step in between. Shown are chemiluminescence detections from representative transfection experiments for each of the panels out of three to nine independent determinations quantified in A (mean Ϯ S.E.). Levels of the co-IP proteins were quantified relative to their total expression amounts (seen in inputs) and then normalized for the immunoprecipitated levels of the indicated PIKfyve construct relative to PIKfyve WT , which was scored as 100%. A strong triple interaction was observed with PIKfyve WT , PIKfyve K1831E , and PIKfyve ⌬FYVE . The association is independent of whether PIKfyve carries GFP-HA or HA tags, but note the different electrophoretic mobility due to the different tag size.
sions. First, impeded binding to the ArPIKfyve-Sac3 subcomplex is associated with markedly reduced lipid kinase activity of the mutants (supplemental Fig. 2 and Fig. 3A). We have to point out, however, that in addition to reduced levels of bound ArPIKfyve-Sac3 subcomplex, resulting in kinase inhibition (12,14,21), conformational changes due to the deletions might also contribute to the drastic loss of the lipid kinase activity and could not be ruled out. Second, if the mutants fail to bind ArPIKfyve-Sac3 with efficiency as high as that of PIKfyve WT , they are unable to trigger vacuolar defects despite drastically reduced (Ͼ30-fold) lipid kinase activity and proper localization to PtdIns(3)P, determined by the intact FYVE domain.
Sac3 WT Exacerbates the Vacuolar Defects by PtdIns(3,5)P 2deficient PIKfyve Mutants with Intact Sac3 Binding-The above result that expression of PIKfyve K1831E⌬Cpnϩ , in contrast to PIKfyve K1831E , is not dominantly interfering suggests that associated ArPIKfyve-Sac3 and particularly the PtdIns(3,5)P 2 -hydrolyzing activity of Sac3 contribute a great deal to the dominant-negative effect of the PtdIns(3,5)P 2 -deficient point mutants on the endomembrane morphology. As we elaborated previously, the dominant-negative phenotype of kinase-deficient PIKfyve K1831E is a complex, gradually developing process that affects different endosomal types depending on the duration of expression (8,18). At earlier stages of COS cell transfection (9 -15 h), the PIKfyve K1831E -positive vesicles are enlarged, but translucent vacuoles are not visible. The latter emerge 15-24 h post-transfection, first at the perinuclear region and then in the whole cell. The vacuoles, with or without PIKfyve K1831E on the limiting membrane, progressively increase in size and decrease in number as a result of fusion (8,18). Therefore, to assess the role of Sac3 activity in induction and progression of the PIKfyve K1831E dominant-negative effect, we examined the phenotypic changes in COS cells triple-transfected with PIKfyve K1831E , ArPIKfyve WT , and Sac3 in either phosphatase-active (Sac3 WT ) or -deficient forms (Sac3 D488A ), both forming PAS complexes with similar efficiencies (Fig. 1, A  and B). Cells were monitored by immunofluorescence microscopy at two time points: 10 -12 and 22-24 h post-transfection. Intriguingly, the cell phenotype under triple expression of eGFP-PIKfyve K1831E , Myc-ArPIKfyve WT , and HA-Sac3 WT was markedly different compared with that under expression of PIKfyve K1831E alone. Thus, the early phase of endosome vesicle swelling seen under single eGFP-PIKfyve K1831E expression was not manifested (Fig. 5A). Rather, the PIKfyve K1831E / ArPIKfyve WT /Sac3 WT -transfected cells displayed large translucent vacuoles within the whole cell as early as 10 -12 h post-transfection, which persisted 24 h post-transfection (Fig.  5, A and B). By contrast, cells expressing eGFP-PIKfyve K1831E , Myc-ArPIKfyve WT , and HA-Sac3 D488A displayed neither dilated endosomes at the early phase nor translucent vacuoles in the later phase to a substantial degree (Fig. 5, A and B). Noteworthy, no significant Sac3-related phenotypic changes were Washed IPs were subjected to assays for lipid kinase activity or autophosphorylation. Shown are autoradiograms of a plate with TLC-separated radiolabeled lipids and of the autokinase reaction resolved by SDS-PAGE and transferred onto a membrane, subsequently immunoblotted (IB) with anti-HA antibodies. Shown is a representative experiment of five to eight experiments with similar results. The lipid kinase activity is quantified by radioactive counting of the scraped silica and is shown in Fig. 3A. B, COS7 cells were transfected with cDNAs of eGFP-HA-PIKfyve K1831E or eGFP-HA-PIKfyve K1831E⌬Cpnϩ . Twenty hours post-transfection cells were treated with or without wortmannin(100nM/20min/37°C,conditionsthatinCOScellsdonotinducewortmannindependent aberrant morphology; see Ref. 18) and then fixed in 4% formaldehyde. Fluorescence and phase-contrast images of transfected cells were captured by a Nikon Eclipse TE200 microscope (Apo 60ϫ/1.40 Ph3DM oil objective) and processed as detailed under "Experimental Procedures." Shown are typical images of the defective vacuolar phenotype induced by PIKfyve K1831E (panels a and b) seen if eGFP-PIKfyve K1831E was coexpressed with Sac3 WT or Sac3 D488A in the absence of ArPIKfyve WT (Fig. 5B).
As a further test of our hypothesis for Sac3 functioning as an active phosphatase in the PAS complex, we examined the effect of Sac3 WT versus Sac3 D488A on cell morphology under expres-sion of the PIKfyve K2000E point mutant. As opposed to Lys-1831 or Lys-1999, Lys in position 2000 was previously characterized to be partially engaged in PtdIns(3,5)P 2 coordination, resulting in only an ϳ40% decreased in vitro PtdIns(3,5)P 2 synthesis by PIKfyve K2000E (17). At this level of activity, PIKfyve K2000E , FIGURE 5. ArPIKfyve WT -Sac3 WT exacerbates, whereas ArPIKfyve WT -Sac3 D488A prevents the endomembrane defects by Sac3-binding PIKfyve mutants defective in PtdIns(3,5)P 2 synthesis. A, COS7 cells were triple-cotransfected with cDNAs of eGFP-HA-PIKfyve K1831E , Myc-ArPIKfyve WT , and either HA-Sac3 WT or HA-Sac3 D488A . Twenty-four hours post-transfection cells were fixed in 4% formaldehyde and stained consecutively for Sac3 (anti-Sac3 IgG) followed by Alexa350 anti-rabbit secondary antibody) and ArPIKfyve (anti-Myc monoclonal antibody followed by Alexa568 anti-mouse secondary antibody). Fluorescence and phase-contrast images were captured by a Nikon Eclipse TE200 microscope (Apo 60ϫ/1.40 Ph3DM oil objective) using three standard filter sets; green for eGFP, red for Alexa568, and blue for Alexa350. Note the presence of multiple vacuoles (panels a-d) under coexpression of enzymatically active Sac3 WT  unlike PIKfyve K1831E or PIKfyve K1999E , does not induce vacuolar defects in transfected COS cells (17). Remarkably, coexpression of Sac3 WT and ArPIKfyve with PIKfyve K2000E triggered profound cell vacuolation, whereas coexpression of Sac3 D488A -ArPIKfyve did not substantially affect the PIKfyve K2000Edependent cell morphology. Quantitation of the PIKfyve K2000Einduced vacuolar phenotype due to Sac3 WT versus Sac3 D488A from two independent transfection experiments is presented in Fig. 5C. Taken together, the results illustrated in Figs. 4 and 5 are consistent with the conclusion that Sac3 functions as a PtdIns(3,5)P 2 phosphatase in the context of the PAS complex.
ArPIKfyve Ct That Disassembles the PAS Complex Alleviates the Phenotypic Defects by PIKfyve K1831E -As revealed recently, ArPIKfyve homomeric interactions mediated by the ArPIKfyve C-terminal domain scaffolds the PAS complex (14). Therefore, as a final verification of our hypothesis that Sac3 functions as a phosphatase when associated with PIKfyve, we examined if disassembly of the PAS complex by ArPIKfyve Ct (14) alleviates the phenotypic defects induced by PIKfyve K1831E in transfected COS cells. As illustrated in Fig. 5D, expressed Myc-PIKfyve K1831E was significantly less potent in triggering formation of aberrant vacuoles if cells coexpressed ArPIKfyve Ct . Quantitation of 2 separate experiments counting ϳ250 transfected cells revealed that coexpressed ArPIKfyve Ct diminished the number of vacuolated cells due to PIKfyve K1831E by 42 Ϯ 4%. These data further indicate that the dominant phenotype of aberrant swollen vacuoles is due to two superimposed events; first, disrupted PIKfyve-catalyzed PtdIns(3,5)P 2 synthesis at endosomal microdomains determined by the PIKfyve FYVE finger and, second, Sac3-dependent hydrolysis localized at the PtdIns(3,5)P 2 synthetic sites through association of the ArPIKfyve-Sac3 subcomplex with PIKfyve.

DISCUSSION
We have recently reported that in native mammalian cells, the PtdIns(3,5)P 2 -metabolizing enzymes PIKfyve kinase and Sac3 phosphatase are organized in a common complex, the PAS core, that includes also the PIKfyve regulator ArPIKfyve (9,14). Heterologous expression in mammalian cell systems had further revealed that the three proteins are both necessary and sufficient to form and maintain a stable PAS complex and that ArPIKfyve and Sac3 are mutually dependent for an efficient association with PIKfyve (14). The integrity of the PAS core complex has been found critical for PIKfyve activation, indicating an unusual requirement for the Sac3 phosphatase in PtdIns(3,5)P 2 synthesis (14). Thus, given the role of Sac3 role in PtdIns(3,5)P 2 biosynthesis and the presence of a PIKfyve-independent ArPIKfyve-Sac3 subcomplex, an outstanding question awaiting clarification is whether the formation of the PAS complex is permissible with Sac3 functioning as a phosphatase or whether its hydrolyzing activity is inhibited upon association. The answer to this question becomes even more prominent in light of the documented ϳ3.5-fold decrease of cellular PtdIns(3,5)P 2 in a Sac3 knock-out mouse model (28), thus raising doubts of whether intracellular Sac3 functions as a PtdIns(3,5)P 2 phosphatase or whether it just facilitates the PtdIns(3,5)P 2 synthetic arm. To this end, in this study we initiated a detailed biochemical characterization of the Sac3 mode of interacting with ArPIKfyve and PIKfyve and used the well established phenomenon of an aberrant vacuolar phenotype as a functional readout for reduced PtdIns(3,5)P 2 -localized levels (7,8,13,(17)(18)(19) and, hence, Sac3 activity. Our observation that elimination of the Cpn60_TCP1 domain in PIKfyve resulted in nearly a total loss of bound ArPIKfyve and Sac3 is consistent with the notion that this region plays a major role in associating the ArPIKfyve-Sac3 subcomplex (Fig. 3, A-C). In contrast, neither the PIKfyve kinase activity nor the Sac3 phosphatase activity played a role in the PAS core formation or stability, as evidenced by the wild-type associations of the activity-deficient point mutants PIKfyve K1831E and Sac3 D488A (Figs. 1 and 3, A  and F). Intriguingly, deletion of the ArPIKfyve-Sac3 binding region mitigated the potency of the kinase-dead PIKfyve K1831Etruncated mutant to dominantly interfere with endomembrane homeostasis and trigger vacuolar formation even when coexpressed with Sac3 WT and/or ArPIKfyve WT , consistent with reduced Sac3 access to, and turnover of PtdIns(3,5)P 2 (Fig. 4). Concordantly, PIKfyve K1831E , with an intact Cpn60_TCP1 domain and wild-type binding to ArPIKfyve-Sac3 (Fig. 3A), induced drastically exacerbated vacuolar defects when coexpressed with ArPIKfyve WT -Sac3 WT but a less severe phenotype if coexpressed with ArPIKfyve WT -Sac3 D488A (Fig. 5, A and B). Finally, the PIKfyve K1831E -dependent vacuolar defects were alleviated by disassembly of the ternary complex with ArPIKfyve Ct (Fig. 5D). These data are consistent with the conclusion that endogenous Sac3, assembled in the PAS core, is an active PtdIns(3,5)P 2 phosphatase. Thus, our results provide the first experimental evidence that the intracellular PAS core complex relays two opposing enzymatic activities: PtdIns(3,5)P 2 synthesis (14) and PtdIns(3,5)P 2 turnover (this study).
Of particular importance in this study is the characterization and quantitative evaluation of the triple associations with a battery of deletion and point mutants (Figs. 1-3). These data lend new mechanistic insight allowing us to propose an in-depth model of the PAS complex interacting domains and their significance for the enzymatic activities (Fig. 6). In addition to the major role of Cpn60_TCP1/CH in the association with the ArPIKfyve-Sac3 subcomplex discussed above, this model is based on several other observations and conclusions as follows.
(i) Sac3 and ArPIKfyve associate with each other through their C termini (Fig. 1, A-D). (ii) Sac3 C terminus associates with at least two ArPIKfyve copies (hence, ArPIKfyve n -Sac3, where n Ն 2), all of them interacting through their C termini (Fig. 1E). (iii) A stable subcomplex made of ArPIKfyve WT and Sac3 C terminus or vice versa does not efficiently bind PIKfyve, indicating both ArPIKfyve and Sac3 N termini stabilize the PAS core complex (Fig. 2). (iv) The PIKfyve FYVE finger is irrelevant in the PAS complex formation (Fig. 3, A and B). (v) A PIKfyve fragment spanning residues 1-1261 that includes the entire Cpn60_TCP1 and CH homology domains has only ϳ25% of the wild-type binding to ArPIKfyve-Sac3. In fact, neither of the two overlapping PIKfyve halves reaches half of the wild-type interaction with the ArPIKfyve-Sac3 WT subcomplex, conceivable with conformational changes to expose other PIKfyve interacting domains (Fig. 3, A, D, and G). (vi) A PIKfyve fragment downstream of Cpn60_TCP1, including the kinase domain, interacts with the ArPIKfyve-Sac3 subcomplex, whereas a fragment with a deleted kinase domain, ⌬Kin, exhibits reduced binding to ArPIKfyve-Sac3, consistent with additional contact sites for ArPIKfyve n -Sac3 binding within the kinase domain (Fig. 3). (vii) Interaction sites between the Sac3 N terminus and PIKfyve are likely to be exposed after the ArPIKfyve n -Sac3 docking to PIKfyve because Sac3 WT and PIKfyve WT do not interact under pairwise analyses (14). Thus, taking into consideration this experimental evidence, we propose a model whereby an ArPIKfyve n -Sac3 subcomplex, formed by the C termini of each subunit, docks at the Cpn60_TCP1/CH homology region of PIKfyve (Fig. 6). We suggest that this induces a conformational change in PIKfyve to allow additional PAS-stabilizing interactions that engage a significant portion of the PIKfyve C-terminal region, including the spectrin repeats and kinase domain, on one hand, and the N termini of ArPIKfyve n -Sac3, on the other. Because the triple association activates PIKfyve (14), recruited Sac3 in the PAS complex ensures the PtdIns(3,5)P 2 homeostatic control by rapid turnover counterbalancing locally elevated PtdIns(3,5)P 2 .
Two recent studies have now confirmed the triple assembly of mammalian PIKfyve, ArPIKfyve, and Sac3 with the respective yeast orthologs Fab1, Vac14, and Fig4 (26,30). Despite some discrepancies between the two reports, these new observations are of considerable significance as they establish evolutionary conservation of the PAS ternary assembly, as we previously predicted (7). Thus, based on biochemical data for the triple associations in yeast (26) and those reported by us in mammalian cells (9,14) or presented herein, it could be concluded that the organization of mammalian and yeast PAS complexes share many similar characteristics. These include mutual dependence of ArPIKfyve/Vac14 and Sac3/Fig4 for a productive association with PIKfyve/Fab1, no direct interaction between PIKfyve/Fab1 and Sac3/Fig4, requirement for the PIKfyve/Fab1 chaperonin-like domain in the ArPIKfyve/ Vac14-Sac3/Fig4 interaction (Fig. 3), ArPIKfyve/Vac14 homodimerization, independence of the ternary assembly from the enzymatic activities of either the kinase or the phosphatase (Figs. 1 and 3), impeded PIKfyve/Fab1 activity if Cpn60_TCP1 domain is mutated (Fig. 4), and membrane localization of the ternary complex via the PIKfyve/Fab1 FYVE finger domain. However, certain differences are also evident, the most apparent one being the affinity of the interactions. Although affinity constants are yet to be determined, the triple complex in mammalian cell systems might be of higher affinity. This conclusion is corroborated by the biochemical detection of the PAS complex with the mammalian endogenous proteins under stringent conditions of RIPA buffer detergents (9), whereas that with the FIGURE 6. Model for the interacting domains of the three proteins and regulated enzymatic activities in the PAS complex. PIKfyve associates with PtdIns(3)P-enriched endosome membranes via its FYVE finger, which is independent of the ArPIKfyve-Sac3 subcomplex. Without bound ArPIKfyve-Sac3, PIKfyve relays submaximal activity due to kinase-unfavorable conformation. The ArPIKfyve n -Sac3 complex, formed through an association of the C termini of an ArPIKfyve dimer (or higher-order homooligomer; hence ArPIKfyve n , where n Ն 2) and the C terminus of a Sac3 monomer, docks to the Cpn60_TCP1 and the CH homology domains of PIKfyve. This induces conformational changes in PIKfyve to uncover binding sites at the catalytic domain, which associate with the N termini of ArPIKfyve n and Sac3. These interactions stabilize a productive PAS core and allow PIKfyve to acquire an "activated" conformation. This transiently increases local synthesis of PtdIns(3,5)P 2 that serves as recognition sites for PtdIns(3,5)P 2 effectors. The local increase in PtdIns(3,5)P 2 is counterbalanced by Sac3, which when incorporated in the PAS core, retains its activity for PtdIns(3,5)P 2 hydrolysis. Neither the Sac3 nor the ArPIKfyve N terminus is dispensable for a productive PAS core complex even though a stable subcomplex via their C termini is formed. The PAS complex could also be formed in the cytosol and subsequently recruited to PtdIns(3)P on membranes.
yeast native proteins remains to be seen. Even under genetic manipulations, the biochemical demonstration of the yeast triple complex requires as much as 13 tandem epitope-tag copies on Fab1 and mild conditions of a single detergent at a low concentration (26). Therefore, perhaps the identification of the yeast triple assembly was initially unsuccessful (31)(32)(33) and lagged behind that in mammalian cells (9,21).
The demonstration that the PAS core relays not only PIKfyve-catalyzed synthesis but also Sac3-catalyzed breakdown of PtdIns(3,5)P 2 is indicative of a pivotal need for tight control of the PtdIns(3,5)P 2 homeostatic mechanism and strict coordination of the antagonistic enzymatic activities. Concordantly, in multicellular organisms such as Drosophila melanogaster or Caenorhabditis elegans, PtdIns(3,5)P 2 is essential for life as evidenced by the embryonic lethality of the PIKfyve-null mutants, in parallel with undetectable PtdIns(3,5)P 2 levels (29,34). A still unresolved question is how important PtdIns(3,5)P 2 is for the life of mammals given that PIKfyve-null mice are still unavailable. Transgenic models of Sac3 and ArPIKfyve are reported, but they do not provide a coherent relationship between the reduced PtdIns(3,5)P 2 and lethality. Thus, ArPIKfyve knockout mice displaying 50% of the normal PtdIns(3,5)P 2 intracellular levels die 1-2 days after birth, whereas the Sac3 mouse model lives to the age of ϳ6 weeks with only ϳ28% of normal PtdIns(3,5)P 2 (28,35). As ArPIKfyve and Sac3 are expected to affect the same subcellular PtdIns(3,5)P 2 pool produced by PIKfyve, this discrepancy is currently unclear. Additional functional inputs by ArPIKfyve (36) could not be excluded.
In conclusion, the data herein provide new mechanistic and functional insight about the domain organization of the PAS complex and its consequence to Sac3 function as a phosphatase. Perturbation of Sac3 phosphatase activity favors PtdIns(3,5)P 2 synthesis over turnover, an observation that might be of therapeutic interest for processes that depend on elevated PtdIns(3,5)P 2 (4,12,37). An important challenge for future studies is the nature of the extracellular stimuli and molecular mechanisms that coordinate the kinase and phosphatase activities within the same complex to assure proper performance of endosomal operations.