A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

Vac14 is ubiquitously expressed in mammalian cells and tissues. Lysates derived from indicated cell lines (A, 200 μg of protein) or mouse tissues (B, 120 μg of protein) were analyzed by SDS-PAGE (6% acrylamide) and immunoblotting with anti-hVac14 antibodies. Shown are chemiluminescence detections of representative immunoblots out of two to three independent experiments for the indicated cell lines or tissues yielding similar results. st., standard; sk, skeletal; mam., mammary; WB, Western blot.

Ectopic expression of Vac14 triggers endogenous protein expression. (A) HEK293 cells first transfected with the indicated pEGFP constructs were cotransfected with the human vac14 siRNA duplexes 5 h later as indicated. Cell lysates, collected 72 h posttransfection, were resolved along with an HA-hVac14 molecular size marker by SDS-PAGE (6% acrylamide) and were immunoblotted with the indicated antibodies, with a stripping step in between. The arrowheads in the upper panel depict the mobility of expressed EGFP-hVac14 or endogenous hVac14 and their selective elimination by Vac14 siRNAs. The arrowheads in the lower panel depict equal expression of EGFP-hVac14 and control EGFP-p40, yet only the former increases endogenous hVac14 levels (upper panel). (B) COS-7 cells were transfected with the indicated pEGFP constructs. Cell lysates, collected 40 h posttransfection, were resolved by SDS-PAGE (12% acrylamide) and immunoblotted with the indicated antibodies, with a stripping step in between. WB, Western blot.

Vac14 and PIKfyve partition similarly between cytosol and membranes and cofractionate in equilibrium gradient sedimentation. (A) HEK293 cells were fractionated into cytosol (Cyt) and total membranes (TM). Aliquots of the fractions along with an HA-hVac14 molecular size marker were resolved by SDS-PAGE and immunoblotted with anti-hVac14. The loading in TM versus Cyt is threefold more on the basis of cell number. Shown is a chemiluminescence detection of a representative blot out of three independent fractionations with similar results. (B and C) HEK293 stable cell line (clone 9) transiently transfected with pEGFP-HA-hVac14 and induced to express PIKfyve^WT was fractionated into TM and Cyt. Both fractions were analyzed by immunoblotting with anti-HA to detect expressed EGFP-HA-hVAc14 or HA-PIKfyve as indicated (B). The loading in TM versus Cyt is twofold more on the basis of cell number (B). TM fractions derived from the stably induced and transiently transfected conditions were subjected to equilibrium sedimentation in 30% iodixanol, as described in Materials and Methods. (C) Fractions were collected from the bottom of the gradient. Aliquots were analyzed by SDS-PAGE (6% acrylamide for PIKfyve, hVac14, EGFP-HA-hVac14, and IRAP or 10.5% acrylamide for Rab4) and immunoblotting with the indicated antibodies, with a stripping step in between. The anti-HA blot depicts the sedimentation profile of expressed EGFP-HA-hVac14. Shown are chemiluminescence detections of blots from a representative experiment out of three independent fractionations yielding similar results. WB, Western blot.

Confocal microscopy reveals substantial colocalization of ectopically expressed hVac14 with PIKfyve or MPR. Twenty-four hours posttransfection with pEGFP-HA-hVac14 alone or together with pCMV5-Myc-PIKfyve^WT or pCMV5-Myc-PIKfyve^K1831E, COS-7 cells were fixed in formaldehyde and permeabilized. Expressed Myc-PIKfyve^WT or Myc-PIKfyve^K1831E was detected with anti-myc monoclonal antibody and Texas red-conjugated secondary anti-mouse antibody, whereas CI-MPR was detected with the polyclonal anti-MPR and CY3-conjugated secondary anti-rabbit antibodies. Expression of EGFP-HA-hVac14 was visualized by GFP fluorescence. Cells were viewed in a Zeiss LSM 510 confocal microscope. Merged images of the green and red channel, presented in panels a, e, and i, illustrate a typical expressing and coexpressing cell per condition and depict a substantial area of colocalization of hVac14 with PIKfyve^WT, CI-MPR, or PIKfyve^K1831E (yellow). The boxed areas in panels a, e, and i are enlarged images, the green (b, f, and j) or red (c, g, and k) channels and their respective merge (d, h, and l). Note that the yellow signal appears on the limiting membrane of perinuclear vesicles (representative vesicles are pointed to by arrowheads in panels d, h, and l).

hVac14 protein knockdown renders cells susceptible to vacuolation. HEK293 cells were transfected with siRNA Smart pool directed to human vac14 or cyclophilin A as indicated. Seventy-two hours posttransfection, cells were treated with NH₄Cl (10 mM) for 40 min at 37°C and then observed live by a light microscope (TE200; Nikon) with a 40× Hoffman modulation contrast objective. Shown are images of live cells captured by a SPOT RT Slider camera. NH₄⁺ treatment induced multiple cytoplasmic vacuoles in cells transfected with vac14 siRNAs (seen in 85% ± 10%, mean ± standard errors of the mean; n = 3) but was completely ineffective in cells transfected with cyclophilin A siRNAs.

Modulations in hVac14 protein expression levels affect PIKfyve lipid kinase activity. (A and B) HEK293 cells were transfected with the indicated siRNAs. PIKfyve lipid kinase activity (A) or protein expression levels (B) were assayed 72 h posttransfection as described below. (C to E) HEK293 cell line (clone 9) was transfected or not transfected with pCMV5-HA-hVac14 and then was treated with or without doxycycline to induce HA-PIKfyve^WT protein expression as indicated. PIKfyve lipid kinase activity (C and E) or protein expression levels (D) were assayed 40 h posttransfection or induction. (A, C, and E) Immunoprecipitates of cell lysates prepared with the indicated antibodies or preimmune sera (Pre) were immobilized on protein A-Sepharose beads and, after washings, were subjected to lipid kinase assay as described in Materials and Methods. (E) Samples were first preincubated with or without wortmannin (20 nM, 15 min) and then were subjected to lipid kinase assay. Shown are autoradiograms of the thin-layer chromatography-resolved lipids from representative experiments out of three (A) and six (C) experiments with similar results. (B and D) Lysates derived from cells in panels A and C were analyzed by SDS-PAGE (6% acrylamide) and immunoblotting with the indicated antibodies. Shown are chemiluminescence detections of representative immunoblots out of three to five experiments for each condition. IP, immunoprecipitate.

Modulations in hVac14 protein expression levels alter intracellular PtdIns(3,5)P₂ in ³²P-labeled HEK293 cells. (A) HEK293 cells were transfected with the indicated siRNAs derived from the corresponding sequences of human vac14 or cyclophilin A. Seventy-two hours posttransfection, cells were labeled with [³²P]orthophosphate as described in Materials and Methods. Cell lipids were extracted, deacylated, and coinjected with [³H]GroPIns 4-P, [³H]GroPIns 3-P, and [³H]GroPIns 4,5-P₂ as internal HPLC standards (elution times are indicated by arrows). The elution times of [³²P]GroPIns 3,5-P₂ and [³²P]GroPIns 3,4-P₂ standards (arrows) were determined from parallel HPLC runs. Fractions were monitored for ³H and ³²P radioactivity by an online flow-scintillation analyzer. Shown are HPLC elution profiles of a typical labeling experiment out of five experiments with similar results. (B) Quantitation of results shown in panel A (siRNAs). Also presented is the quantitation of three independent labelings in the HEK293 cell line (clone 9) expressing PIKfyve^WT, cotransfected with a control pEGFP vector or pEGFP-HA-hVac14, and labeled 24 h posttransfection with [³²P]orthophosphate as shown in panel A. For quantitation, the radioactive peaks were first calculated as a percentage of total PI radioactivity and then were expressed as a percentage of the PtdIns(3)P, PtdIns(3,5)P₂, or PtdIns(3,4)P₂ relative level in the corresponding control condition. Data are presented as means ± standard errors of the means.

hVac14 physically associates with PIKfyve. (A and B) PC12 cells, abundant in endogenous PIKfyve and Vac14 (lane 1 in panels A and B), were lysed in RIPA buffer and were immunoprecipitated with the preimmune serum from the Vac14 antibody production, anti-hVac14, and two anti-PIKfyve antibodies against the N terminus and C terminus (anti-PIKfyve_N and anti-PIKfyve_C, respectively) as indicated. Immunoprecipitates were resolved by SDS-PAGE (6% acrylamide) and were immunoblotted with anti-PIKfyve (A) and anti-hVac14 antibodies (B), with a stripping step in between. Shown are chemiluminescence detections of a blot from a representative experiment out of five independent coimmunoprecipitation experiments in this cell type yielding similar results. (C and D) Lysates of an HEK293 cell line (clone 9) induced to express HA-PIKfyve^WT that were immunoprecipitated with anti-hVac14 (affinity purified), anti-HA antiserum, or preimmune serum from the Vac14 antibody production, as indicated, were resolved by SDS-PAGE (6% acrylamide) and were immunoblotted as described for panels A and B. (E) Immunoprecipitates of PC12 cell lysates prepared with anti-hVac14, anti-PIKfyve (against the N terminus), or irrelevant antisera (Non-imm) as well as with the preimmune serum of anti-hVac14 production were immobilized on protein A-Sepharose beads and, after washings, were subjected to lipid kinase assay as described in Materials and Methods. Extracted lipids were resolved by thin-layer chromatography. Shown is an autoradiogram from a representative experiment out of three with similar results. WB, Western blot; Preimm, preimmune; IP, immunoprecipitate.