PMC

Active site mutation of APT1 increases membrane localization of both APT1 and APT2. Cultured astroglia were transfected with the APT1, APT1-M-1, or APT1-M-2 construct. Note that in cells transfected with the APT1 construct, APT1 fluorescence co-localized with that of Na⁺/K⁺ATPase (A, upper row). The cells transfected with APT1-M-2 construct showed increased co-localization with the cell membrane marker (A, lower row). However, in APT1-M-1-transfected cells, the APT1 fluorescence predominantly localized in the cytosol and perinuclear areas (A, middle row). Compared with NIH3T3 cells transfected with wild type APT1 (B, upper row), those transfected with the APT1-M-1 construct showed increased co-localization of APT-2 fluorescence with that of the membrane marker Na⁺/K⁺-ATPase (B, middle row). The cells transfected with APT1-M-2 construct also showed increased co-localization of APT2 immunofluorescence with that of Na⁺/K⁺-ATPase (B, lower row).

Schematic model explaining how dynamic palmitoylation might regulate steady-state membrane localization and function of APT1 and APT2 and those of H-Ras and GAP-43. In this model, APT1 and APT2 undergo palmitoylation in the Golgi by as yet unknown PATs, which facilitates their membrane localization. Membrane-associated APT1 and APT2 then depalmitoylate H-Ras (1) and GAP-43 (2), respectively, detaching them from the cell membrane and promoting translocation to the Golgi where they are re-palmitoylated and translocated to the membrane to manifest their function. Importantly, APT1 also depalmitoylates APT2 (3) by detaching it from the membrane for another cycle of palmitoylation in the Golgi. Following depalmitoylation of APT2, APT1 then catalyzes its own depalmitoylation for translocation to the Golgi to undergo re-palmitoylation as required for membrane localization. However, although in this model APT1 catalyzes depalmitoylation of APT2, APT2 does not depalmitoylate APT1.

Co-localization of APT1 and APT2 immunofluorescence with that of Na⁺/K⁺ATPase. Quantitation of APT1 or APT2 immunofluorescence with Na⁺/K⁺ATPase was performed using AIM 4.2 software (Carl Zeiss) to identify co-localization in the overlapping areas of the image. Insets show co-localization of APT1 (A, panel i) and APT2 (A, panel ii) with the cell membrane marker Na⁺/K⁺ATPase. There were no co-localizations of APT1-M-1 (A, panel iii) and APT2-M-1 (A, panel iv) with the cell membrane marker. Pearson's correlation coefficient (Rr) of APT1 or APT2 is greater than 0.75 in the overlapping areas (mean from seven individual measurements) (B). Moreover, there were no significant correlations of APT1-M-1 and APT2-M-1 with cell membrane marker, Na⁺/K⁺ATPase.

Suppression of Apt1 and Apt2 palmitoylation by bromopalmitate. Suppression of Apt1 palmitoylation by bromopalmitate dose-dependently increased the level of cytosolic Apt1 (A) and reduced its membrane localization (B). Immunocytochemical analyses were performed using Apt1 and Na⁺/K⁺ATPase antibodies, which showed that Apt1 is localized predominantly in the cytoplasm of the cells treated with bromopalmitate (C, lower row), although accumulation of Apt1 is clearly detected on the membrane of the cells treated with DMSO (C, upper row). The cytosolic and membrane fractions from bromopalmitate-treated cells were probed with APT2 antibody. Suppression of Apt2 palmitoylation by bromopalmitate markedly increased the cytosolic Apt2 (D) and decreased its membrane localization (E). Immunocytochemical analyses were performed using Apt2 or Na⁺/K⁺ATPase- antibodies. Note that the Apt2 signal on the cell membrane is virtually abolished in bromopalmitate-treated cells (F, lower row) compared with DMSO-treated control cells (F, upper row).

Effects of shRNA suppression of Apt1 or Apt2 on membrane localization of Apt1 and Apt2. Compared with scrambled shRNA-transfected cells (A, lane 1), those transfected with APT1-shRNA (A, lane 2) had appreciably decreased levels of Apt1 protein. Apt1 knockdown reduced the level of Apt2 in the cytosolic fractions (B) but increased that of Apt2 in the membrane fractions (C). APT2-shRNA transfection markedly decreased Apt2 expression (D, lane 2) compared with that of scrambled shRNA-transfected cells (D, lane 1). Western blot analysis of cytosolic (E) and membrane fractions (F) from APT2-shRNA-transfected cells showed virtually no alteration in Apt1 protein level. Immunocytochemical analysis was performed with either Apt1 or Apt2 and Na⁺/K⁺ATPase antibody. Compared with scrambled shRNA-transfected cells (G, upper row), those transfected with APT1-shRNA had a markedly higher Apt1 signal co-localized with that of Na⁺/K⁺ATPase (G, lower row). However, compared with its scrambled shRNA-transfected cells (H, upper panel), those transfected with APT2-shRNA had very similar Apt1 signal co-localization with that of the membrane marker Na⁺/K⁺ATPase (H, lower row).

Palmitoylation of APT1 or APT2 promotes their membrane localization. Palmitoylation site (Cys-2) was predicted by CSS-Palm 3.0 analysis at the highest stringency in mouse APT1 and APT2 (A, upper two rows) and human APT1 and APT2 (A, lower two rows). Palmitoylated APT1 (B, lane 2) and APT2 (C, lane 2) were readily detectable by ABE assay, although C2S mutation in both APT1 (B, lane 4) and APT2 (C, lane 4) abrogated palmitoylation. Palmitoylation of APT1 (D, lane 1) and APT2 (E, lane 1) were further confirmed by the presence of [¹⁴C]palmitate-labeled APT1 and APT2 rotein bands, which were rendered undetectable by hydroxylamine treatment (D and E, lanes 2). Note that C2S mutation in APT1 (F) and APT2 (G) abrogated membrane ssociation of both APT1 (F, lane 4), and APT2 (G, lane 4). Subcellular localizations of APT1 and APT2 in cells transfected with the APT1-M-1, APT2 or APT2-M-1 construct were analyzed by confocal microscopy. Image data show that although APT1 (H, upper row) and APT2 (I, upper row) were localized to the membrane (arrowheads), the APT1-M-1 (H, lower row) and APT2-M-1 (I, lower row) were predominantly localized to the cytoplasm.

Dynamic palmitoylation of Apt1 and Apt2 regulates steady-state membrane localization of H-Ras and GAP-43, respectively. The cytosolic fractions of cultured astroglia treated with DMSO or Palm B were probed with H-Ras or GAP-43 antibodies as indicated. Note a slight reduction in H-Ras signal in the cytosolic fraction of 1 μm Palm B-treated cells (A). There is, however, no apparent difference in the levels of GAP-43 (B) in the cytosolic fractions of Palm B-treated and control cells. The membrane fractions from astroglial cells treated with DMSO (control) or varying concentrations of Palm B were probed with either H-Ras or GAP-43 antibodies. Note that both H-Ras (C) and GAP-43 (D) levels in the membrane fractions of the cells treated with Palm B were elevated in a dose-dependent manner. Immunocytochemical analysis of cultured astroglia were performed using antibodies to either H-Ras (E) or GAP-43 (F) and Na⁺/K⁺ATPase. Compared with DMSO-treated cells (E and F, upper row, arrowheads), those treated with Palm B showed markedly increased membrane localization of H-Ras (E, lower row, arrowheads) as well as that of GAP-43 (F, lower row, arrowheads).

Membrane localization of APT1 and APT2 in Palm B-treated cells. Palm B treatment in a dose-dependent manner elevated the levels of both Apt1 (A) and Apt2 (B) in membrane fractions of the cells. Palmitoylation status of APT1 (C) and APT2 (D) was checked in cultured astroglial cells expressing APT1 or APT2 in which either palmitoylation or depalmitoylation was inhibited by bromopalmitate and Palm B treatment, respectively. Lysates from 3 × 10⁶ cells for each treatment were used for Western blot analysis. The densitometric quantitation of the protein bands in Western blots from three independent experiments were performed, and the results are presented graphically as the mean ± S.D. Note that Palm B treatment markedly elevated the levels of palmitoylated APT1 (C, lane 3, and bar graph) as well as APT2 (D, lane 3, and bar graph) as compared with their respective controls (C and D, lane 1), although bromopalmitate treatment markedly reduced the level of palmitoylated APT1 (C, lane 2) and APT2 (D, lane 2). Palm B treatment prevents dissociation of both Apt1 (E) and Apt2 (F) from the membrane. Compared with DMSO-treated cells (E and F, upper panels, arrowheads), the Palm B-treated cells had markedly elevated levels of membrane-localized Apt1 (E, lower panel, arrowheads) and Apt2 fluorescence (F, lower panel, arrowheads).