SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis - PubMed (original) (raw)

SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis

Fabienne Soulet et al. Mol Biol Cell. 2005 Apr.

Abstract

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.

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Figures

Figure 1.

Figure 1.

SNX9 interacts in vivo with dynamin-1 and dynamin-2. tTA-HeLa cells were untreated or infected with recombinant adenoviruses encoding HA-dynamin-2ΔPRD, HA-dynamin-2, HA-dynamin-1ΔPRD, and HA-dynamin-1. Total extracts were immunoprecipitated with the anti-HA antibody, 12CA5. The immunoprecipitated proteins were analyzed by immunoblot sequentially using polyclonal α-SNX9 antibodies and anti-HA antibodies.

Figure 2.

Figure 2.

SNX9 interacts directly with dynamin in vitro. The GST fusion proteins, GST-SNX9, GST-SNX9ΔSH3, GST-SH3(SNX9), or GST alone were immobilized on glutathione-Sepharose-beads and incubated with purified dynamin-1, dynamin-1ΔPRD, dynamin-2, or dynamin-2ΔPRD as described in Materials and Methods. Bound proteins were then analyzed by SDS-PAGE and Coomassie blue staining.

Figure 3.

Figure 3.

SNX9 interacts with dynamin and clathrin at the plasma membrane. (A) tTA-HeLa cells were fractionated into membrane enriched (M) or cytosolic fractions (C). Each fraction was separately immunoprecipitated with a monoclonal antidynamin antibody, Hudy-1 or a nonspecific mouse antibody (NS IgG). The immunoprecipitated proteins were analyzed by sequential immunoblot using anti-SNX9 antibody followed by antidynamin polyclonal antibody (4003). (B) Indirect immunofluorescence in HeLa cells of SNX9 (green) shows partial colocalization with dynamin-2 and clathrin. Inset: higher magnification of the indicated area. Red arrows: pits without SNX9; green arrows: pits with only SNX9; yellow arrows: colocalization. SNX9 also colocalizes with the Golgi marker, Golgi p58 protein. Scale bar, 10 μm.

Figure 4.

Figure 4.

SNX9 is transiently recruited to clathrin-coated pits late in coated vesicle formation. Images from time-lapse sequences showing the behavior of (A) CLC-DsRed and EGFP-SNX9 imaged by TIR-FM and WF-EF (see also Supplementary Movies 1 and 2) and (B) EGFP-SNX9 and dynamin1-mRFP imaged by TIR-FM (see also Supplementary Movie 3). Scale bar, 1 μm.

Figure 5.

Figure 5.

SNX9 stimulates dynamin basal GTPase activity. The basal rate of GTP hydrolysis was measured at high salt concentration (150 mM KCl) and 0.5 μM dynamin. (A) The amount of Pi released by dynamin-1 (▴, ▵) or dynamin-2 (▪, □) in the presence (closed symbols) or absence (open symbols) of 1 μM GST-SNX9, is plotted as a function of time. (B) Rate of GTP hydrolysis by dynamin-1 was measured and plotted as a function of increasing concentrations of effector: GST-SNX9 (▴), GST-SNX9ΔSH3 (▵), and GST-SH3(SNX9) (○), GST-amph1 (▪), and GST-SH3(amph) (□). (C) Self-assembly of dynamin-1 was examined by velocity sedimentation at physiological salt concentration (150 mM KCl), followed by SDS-PAGE analysis of the pellet (P) and supernatant (S).

Figure 6.

Figure 6.

SNX9 and liposomes synergistically enhance dynamin's assembly-stimulated GTPase activity. (A) The GTPase activity of dynamin-1 (0.2 μM) was measured alone without (○, dashed line) or with (•) liposomes, or in the presence of 0.2 μM GST-SNX9 (▵), 0.2 μM GST-SNX9 and liposomes (•), 0. 2 μM GST-amph1 (□), or 0.2 μM GST-amph1 and liposomes (▪). Assays were performed as described in Materials and Methods. (B) Rates of GTP hydrolysis for dynamin-1 alone, with liposomes, with SNX9 and with SNX9 and liposomes, were measured in three independent experiments. Data shown are averages ± SD.

Figure 7.

Figure 7.

SNX9 is required for efficient TfnR internalization. HeLa cells were transfected with two different siRNA constructs, one targeting specifically SNX9 (siRNA-SNX9) and another nonsilencing (siRNA control). The inset is a Western blot showing that SNX9 expression and not dynamin-2 expression is selectively reduced in siRNA-SNX9 treated cells. After siRNA treatment, single round internalization assays show an inhibition of Tfn internalization with the siRNA-SNX9 measured 72 h after transfection with control (•) or SNX9 (○) siRNAs. The percentage of total surface bound Tfn internalized during incubation at 37°C is plotted as a function of time. Data are averages ± SD from three independent experiments.

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