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 into each domain as indicated. (B) Amino acid sequence alignment of a conserved area in the CCT and CCR domains of several Fab1 orthologues: mm, Mus musculus; Hs, Homo sapiens; Sc, S. cerevisiae; Af, Aspergillus fumigatus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. The asterisk (*) denotes conserved residues, whereas the colon (:) and the period (.) indicate near-identical or similar residues, respectively. Enclosed residues were mutated as indicated above. (C) Cellular levels of PtdIns(3,5)P₂ as a percentage of total PtdIns in cells expressing the indicated Fab1 mutants. (D) Temperature-sensitive growth of fab1Δ cells expressing Fab1 mutants. Serial dilutions of cultures grown at 26 or 38°C for three days. (E) Vacuolar morphology of fab1Δ cells expressing the designated fab1 mutants. Cells were labeled with FM4-64 to demarcate the vacuolar limiting membrane. The FM4-64 and DIC images were merged as a grayscale palette. Bar, 5 μm.

Behavior of the FYVE domain of Fab1. (A) Wild-type (top) or vps34Δ cells (bottom) coexpressing GFP-FYVE^Fab1 and RFP-FYVE^EEA1. (B) fab1Δ (top) or vac7Δ (bottom) cells expressing GFP-FYVE^Fab1. Bar, 5 μm.

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 by SDS-PAGE and probed as indicated. Lysates lacking the cognate epitope for IP were used to ensure the specificity of each coIP. Input lanes represent 10% of the total material used for each IP. (A) IP from whole cell lysates with anti-HA (left) or anti-Myc (right) antibodies and probed with the corresponding antibodies as shown. (B) Cell fractionation into a cytosolic (C) and a membrane/pellet fraction (M). (C) Cytosolic (left) and membrane fractions (right) were incubated with anti-HA antibodies for IP. (D) Cells lysates were prepared from diploid cells expressing HA and Myc-tagged alleles of Fig4 (left) or from diploid cells expressing alleles of Fab1 tagged with HA and Myc (middle). IPs were done with anti-Myc antibodies. Whole cell lysates were made from cells expressing Vac14-FLAG and transformed with either empty vector (−) or with HA-Vac14 (+; right). IP was done with anti-HA antibodies.

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-GroL^T/I domain (A) or from fab1Δ fig4Δ Vac14-FLAG cells expressing empty vector or HA-GroL (B). IPs were separated by SDS-PAGE and probed for HA-GroL, Vac14-FLAG, and Fig4 as described previously. Inputs represent 10% of total material used during the IP. (C) fab1Δ VAC14-GFP cells expressing HA-ALP, HA-GroL-ALP, or HA-HA-GroL^T/I-ALP and labeled with FM4-64. FM4-64 and corresponding DIC images were merged. Line plot analysis was performed as described in Figure 2. Bar, 5 μm. (D) Quantification of V/C of Vac14-GFP signal as described in Figure 2. (E) Coomassie staining of purified bacterially expressed recombinant GST, GST-Vac14, GST-GroL, T7-Vac14-HIS₆ and T7-Fig4-HIS₆. Amount loaded represents ∼20% of input samples for binding reactions. Small bars on the right indicate T7-Vac14-HIS₆ and T7-Fig4-HIS₆ and the asterisk (*) point to degradation products or contaminating proteins. (F) In vitro interaction between Vac14, Fig4, and the GroL-like region of Fab1. Left, GST and GST-Vac14 coupled to glutathione agarose beads were incubated with T7-Vac14-HIS₆ or T7-Fig4-HIS₆. Right, binding reactions between GST alone or GST-GroL fusion protein coupled to glutathione beads and incubated with T7-Vac14-HIS₆ or T7-Fig4-HIS₆. Blots were probed with monoclonal anti-T7 antibodies.

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 of the GFP and FM4-64 signal is shown for each panel. A line 25–40 pixels in length and 5 pixels wide was drawn from the cytosol to the lumen. FM4-64 peak demarcates the vacuole limiting membrane. Fluorescence is normalized for both channels against the highest fluorescence value acquired. C, cytosolic side; V, vacuolar lumen side; dotted line, vacuolar membrane. Bar, 5 μm. (B) Quantification of the vacuole-to-cytosol ratio (V/C) of the Fab1-GFP fluorescence signal. Fluorescence intensity readings were taken by averaging the signal on >25% of the cytosolic area and >50% of the vacuole membrane for each cell. The V/C ratio for n > 20 and its SD are illustrated. Statistical analysis was performed using unpaired, two-tailed Student's t test.

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 DIC images. Line plotting of Vac14-GFP and FM4-64 fluorescence was done as described in Figure 2. Bar, 5 μm. (B) Quantification of V/C for Vac14-GFP and statistical analysis were as described in Figure 2.

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 Fab1 (top right), from fig4Δ cells with or without Myc-tagged Fab1 (bottom left) or from vac7Δ cells with or without Fab1-Myc (bottom right). IPs were separated by SDS-PAGE and probed accordingly and as described in Materials and Methods. Input lanes depict 10% of total protein used for each IP experiment. The asterisk (*) denotes degradation products of Fab1-myc. (B) Cellular levels of PtdIns(3,5)P₂ as a percentage of total PtdIns in atg18Δ fig4Δ cells transformed with an empty vector or vectors expressing ATG18, FIG4, or fig4-1. Bars represent SE, n = 3. (C) IPs using anti-HA antibodies against Vac14-HA from whole cell lysates derived from atg18Δ fig4Δ cells transformed with either empty vector, FIG4 or fig4-1. Samples were handled and probed as described before.

The Fab1 complex model. Fab1 is recruited to the vacuolar membrane by binding to PtdIns(3)P through its FYVE domain. This localization is independent of Vac14 and Fig4 and is likely in dynamic exchange with the cytosol. Vac14 and Fig4 probably form a cytosolic subcomplex that subsequently binds to the CCT and CCR domains Fab1 (the GroL-like region). Vac14 and Fig4 are mutually dependent for interaction with Fab1. Conceivably, the Fab1 complex is induced and/or stabilized by a vacuolar signal. Although Fab1 and Fig4 regulate PtdIns(3,5)P₂ lipid levels, the molecular mechanisms that coordinate the kinase and phosphatase activities are currently unknown.