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. 2015 Apr 13;209(1):97-110.
doi: 10.1083/jcb.201408027.

Spatiotemporal Control of Phosphatidylinositol 4-phosphate by Sac2 Regulates Endocytic Recycling

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Free PMC article

Spatiotemporal Control of Phosphatidylinositol 4-phosphate by Sac2 Regulates Endocytic Recycling

FoSheng Hsu et al. J Cell Biol. .
Free PMC article

Abstract

It is well established that the spatial- and temporal-restricted generation and turnover of phosphoinositides (PIs) by a cascade of PI-metabolizing enzymes is a key regulatory mechanism in the endocytic pathway. Here, we demonstrate that the Sac1 domain-containing protein Sac2 is a PI 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate in vitro. We further show that Sac2 colocalizes with early endosomal markers and is recruited to transferrin (Tfn)-containing vesicles during endocytic recycling. Exogenous expression of the catalytically inactive mutant Sac2C458S resulted in altered cellular distribution of Tfn receptors and delayed Tfn recycling. Furthermore, genomic ablation of Sac2 caused a similar perturbation on Tfn and integrin recycling as well as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway.

Figures

Figure 1.
Figure 1.
Sac2 is a PI 4-phosphatase that specifically hydrolyzes PI(4)P. (A) Mouse tissue lysates were normalized and analyzed by Western blot with specific anti-Sac2 antibody. WBL, whole mouse brain lysate. (B) Different cell lines were harvested and analyzed by Western blot with anti-Sac2 and anti-tubulin antibodies. (C, left) PI phosphatase assay with recombinant Sac2 proteins purified from insect cells. (right) SDS-PAGE analysis of the purified Sac2 samples. (D) Quantification of phosphatase assays shown in C. (E, left) Phosphatase assay with endogenous Sac2 immunopurified by anti-Sac2 antibody. (right) Western blot with anti-Sac2 of the purified protein samples. (F) Quantification of the assay shown in E. Data are from three replicate experiments (mean ± SEM).
Figure 2.
Figure 2.
Sac2CS mutant localizes to punctate structures. (A) Schematic diagram of various constructs used in B. The C458S catalytically inactive mutation is denoted with an asterisk. (B) Confocal images of N2A cells that were transfected with GFP-Sac2, GFP-Sac2CS, or various Sac2 truncation plasmids containing an N-terminal GFP tag. (C) N2A cells were first transfected with GFP-Sac2CS for 24 h and then subjected to phenylarsine oxide (PAO; 20 µM) treatment for 10 min at 37°C. Bars, 5 µm.
Figure 3.
Figure 3.
Sac2 localizes to early and recycling endosomes. (A–E) Colocalization of either GFP- or mCherry-Sac2CS with various endocytic markers. A single plane of spinning disk confocal microscopy is shown. (F) Colocalization analysis of GFP-Sac2CS with Tfn. N2A cells were transfected with GFP-Sac2CS and were treated with Alexa594-Tfn for 15 min before fixation for imaging. Bars, 5 µm. (G) Colocalization analysis of Sac2CS with various endocytic markers. The mean values of the Pearson’s correlation coefficient from three cells and the SEM are shown.
Figure 4.
Figure 4.
Expression of Sac2CS mutant perturbs TfnR distribution and Tfn recycling. (A) Western blot analysis of biotin surface-labeled TfnR. (B) Densitometry analysis of the Western blot images shown in A. Data are from three replicate experiments (mean ± SEM). (C) Flow cytometry analysis of Tfn recycling in N2A cells expressing GFP (control) or GFP-Sac2CS. Data are from three replicate experiments (n = 5,000 in each time point; mean ± SEM). *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
Defects of Tfn recycling in Sac2 null cells. (A) Western blot analysis of biotin surface-labeled TfnR. (B) Quantification was performed as in Fig. 4 B. Values are normalized to WT cells (surface/total). Data are from three replicate experiments (mean ± SEM). (C) Flow cytometry analysis of Tfn recycling in WT and Sac2 null N2A cells. Data are from three replicate experiments (n = 5,000 in each time point; mean ± SEM). (D) Representative images of Tfn recycling at the indicated times points. (bottom) Cells transfected with Flag-Sac2 (green). Bars, 10 µm. *, P < 0.05; **, P < 0.01.
Figure 6.
Figure 6.
Defects of integrin recycling in Sac2 null cells. (A) Flow cytometry analysis of surface integrin in WT (black) and ΔSac2 (red) cells. (B) Quantitative analysis of the mean intensity shown in A. Data are from three replicate experiments (n = 10,000; mean ± SEM). (C) Western blot probed with anti–β1 integrin, anti-tubulin, and anti-Sac2 of cell lysates prepared from WT and Sac2 null cells. (D) Intracellular distribution of β1 integrin in WT and ΔSac2 cells. The mean fluorescence intensity in nonpermeabilized and permeabilized cells and the SEM are shown in the right panels (n = 5). (E) Recycling assay of β1 integrin. Bars, 10 µm. The mean recycled fluorescence intensity in nonpermeabilized and permeabilized cells and the SEM are shown in the right panels (n = 5). **, P < 0.01.
Figure 7.
Figure 7.
Tfn-containing vesicles are positive for PI(4)P in Sac2 null cells. (A) Colocalization assay of GFP-2xPHFAPP1 (green) with Alexa647-Tfn. (B) Pearson’s correlation analysis of the colocalization in A. (C) Colocalization assay of GFP-PHOSBP (green) with Alexa647-Tfn. (D) Pearson’s correlation analysis of the colocalization in C. Error bars represent SEM. mCherry-Sac2 was pseudo-colored in blue. Bars, 5 µm. *, P < 0.05; **, P < 0.01.
Figure 8.
Figure 8.
Sac2 deletion delays cell migration. (A) Wound healing assay of ΔSac2 cells. Dashed lines measure the distance of the wound. FITC images are shown to indicate transfected cells. Bars, 20 µm. (B) Quantification of the distance traveled after 48 h as measured by the dashed lines. Data are from three replicate experiments (mean ± SEM; **, P < 0.01).
Figure 9.
Figure 9.
Crystal structure of the hSac2 domain of Sac2. (A) Ribbon diagram of the overall structure of the hSac2 domain. The hSac2 domain consists of a core of two perpendicularly apposed β sheets (pink) with a C-terminal α helix (blue). (B) Ribbon diagram of the hSac2 dimer. (inset) A zoom-in view showing the four pairs of main chain hydrogen bond formed between the two β1 strands within each monomer. (C) Western blot showing Flag-Sac2 1–798 coimmunoprecipiting with GFP-Sac2 1–798 but much reduced with GFP-Sac2 1–567.
Figure 10.
Figure 10.
Model for Sac2 function during endosomal recycling. The recycling process is dependent on the proper control of PIs. Initial stage of endocytosis requires the hydrolysis of PI(4,5)P2 to PI(4)P via PI 5-phosphatases, such as OCRL. Sac2 is then recruited to endocytic intermediates to hydrolyze PI(4)P to phosphatidylinositol, which is a substrate for Vps34 to generate PI(3)P. The spatiotemporal conversion from PI(4,5)P2- to PI(3)P-enriched endosome is essential for endosomal maturation and subsequent recycling events.

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