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. 2006 Feb;17(2):598-606.
doi: 10.1091/mbc.e05-05-0389. Epub 2005 Nov 16.

Phospholipase D2 is required for efficient endocytic recycling of transferrin receptors

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Phospholipase D2 is required for efficient endocytic recycling of transferrin receptors

David Padrón et al. Mol Biol Cell. 2006 Feb.

Abstract

RNA interference-mediated depletion of phospholipase D2 (PLD2), but not PLD1, inhibited recycling of transferrin receptors in HeLa cells, whereas the internalization rate was unaffected by depletion of either PLD. Although reduction of both PLD isoforms inhibits PLD activity stimulated by phorbol 12-myristic 13-acetate, only depletion of PLD2 decreased nonstimulated activity. Cells with reduced PLD2 accumulated a greater fraction of transferrin receptors in a perinuclear compartment that was positive for Rab11, a marker of recycling endosomes. EFA6, an exchange factor for Arf6, has been proposed to stimulate the recycling of transferrin receptors. Thus, one consequence of EFA6 overexpression would be a reduction of the internal pool of receptors. We confirmed this observation in control HeLa cells; however, overexpression of EFA6 failed to decrease the internal pool of transferrin receptors that accumulate in cells previously depleted of PLD2. These observations suggest that either PLD2 is required for a constitutive Arf6-mediated recycling pathway or in the absence of PLD2 transferrin receptors accumulate in recycling endosomes that are not responsive to overexpression of EFA6.

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Figures

Figure 1.
Figure 1.
Expression of phospholipases D1 and D2 is effectively decreased by RNA interference. Two distinct siRNAs complementary to mRNA sequences of each of PLD1 or PLD2, and one siRNA targeting luciferase (control, C), were transfected into HeLa cells. Forty-eight hours later immunoblots with PLD1, panPLD, and tubulin antibodies were performed, and a representative is shown (A). mRNA for each PLD isoform was measured in cells treated with siRNA specific for one isoform by real-time quantitative PCR. The mean with SD (SD) from three experiments is shown normalized to the control (B).
Figure 2.
Figure 2.
PLD2 is required for efficient recycling of transferrin receptors. Transferrin receptors were labeled by indirect immunofluorescence 2 d after transfection with siPLD2 or with a control siRNA (A). Recycling and internalization of transferrin (see Materials and Methods) were also measured in cells treated with siPLD. Recycling and Internalization graphs present the mean and SD of three experiments.
Figure 3.
Figure 3.
Depletion of PLD2 inhibits transferrin receptor recycling in fibroblasts. SV589 cells were transfected with control and siPLD2 oligonucleotides and 72 h later immunoblots with panPLD and tubulin antibodies were performed. A representative blot is shown (A). Recycling of transferrin was measured as in Figure 2; the mean and SD of three experiments are plotted (B).
Figure 4.
Figure 4.
Decreased expression of PLD1 reduces PMA-induced PLD activity but not basal PLD activity, whereas decreased expression of PLD2 decreases both activities. 3H-Labeled lipids were extracted from duplicate samples of cells treated with the reagents indicated and were analyzed by TLC. An autoradiograph of a representative experiment is shown (A). Autoradiographs of three experiments were quantified by densitometry (arrow), and the average values and SD are plotted on the graph (B).
Figure 5.
Figure 5.
Transferrin receptors accumulate abnormally in Rab11-positive endosomes in cells with reduced PLD2 expression. Control and siPLD2-treated cells were labeled with transferrin receptor and Rab11 antibodies (A) or with fluorescent transferrin and EEA1 antibody (B), and confocal micrographs were taken. Most cells in the siPLD2 samples had a prominent perinuclear accumulation of transferrin receptors; typical cells with this phenotype are shown.
Figure 6.
Figure 6.
EFA6 increases the recycling of transferrin receptors. Cells transfected with GFP only (control) or GFP-EFA6 were labeled with Alexa 633 transferrin at 37°C to fill the endocytic recycling pathway, and then the surface population of transferrin was removed. Cells were then incubated for various intervals at 32°C to allow the internalized transferrin to recycle to the cell surface and be released. The mean fluorescence (Alexa 633) of the cell population expressing GFP at each time point was measured by FACS and plotted as percentage of the value measured with no chase. Curves were fit to the data using the curve-fitting software Prism 3.0cx. Values are the means and SEs of four experiments.
Figure 7.
Figure 7.
PLD2 acts downstream of the EFA6 mediated recycling of transferrin receptors. Cells were transfected with siRNA for PLD2 or a control siRNA, and 2 d later they were microinjected with a plasmid encoding GFP-EFA6. Cells were allowed to internalize fluorescent transferrin and were incubated in the absence of transferrin to allow internalized transferrin to recycle. Cells were fixed and stained with antibody to GFP to identify those overexpressing the exchange factor (A). Cells overexpressing EFA6 and treated with siPLD2 did not show the accelerated loss of fluorescent transferrin seen in cells treated with control siRNA. For quantification of transferrin, micrographs were taken using identical settings. Graph (B) shows average pixel intensity and SD of EFA6-expressing cells in control (n = 21) and siPLD2 (n = 15)-treated cells as well as that of neighboring uninjected cells (n = 34 from control samples and n = 14 from siPLD2 samples).
Figure 8.
Figure 8.
Depletion of PLD2 reduces PIP2 production, but overexpression of PIP5KI does not relieve inhibition of transferrin recycling. Cells were treated with siRNAs to PLD1, PLD2, or a control. Cells were labeled with [32P]phosphoric acid and then lipids were extracted and analyzed by TLC. An autoradiogram with duplicate samples of a representative experiment is shown. (A) PIP and PIP2 are indicated by the arrows. The average (±SD) of three experiments quantified by densitometry are plotted as percentage of control and shown in B and C. Cells depleted of PLD2 were microinjected with myc-tagged hPIP5KIβ, labeled with fluorescent transferrin as in Figure 7, and stained with anti-myc antibody (D).

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