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. 2007 Nov 7;2(11):e1144.
doi: 10.1371/journal.pone.0001144.

The redox sensor TXNL1 plays a regulatory role in fluid phase endocytosis

Michela Felberbaum-Corti  1 , Etienne Morel, Valeria Cavalli, Francis Vilbois, Jean Gruenberg
Affiliations

The redox sensor TXNL1 plays a regulatory role in fluid phase endocytosis

Michela Felberbaum-Corti et al. PLoS One. .

Abstract

Background: Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis.

Methodology/principal findings: When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5.

Conclusions/significance: Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TXNL1 and p38MAPK.
(A) Rat liver cytosol (RLC, 30 mg) was precipitated in 30% (NH4)2SO4 (P30). The precipitate was fractionated by gel filtration chromatography on a Superose 12 column, and fractions analyzed by western blotting. The lower panel shows the densitometric scan of the gels in the upper panel (red squares: TXNL1; blue triangles: p38MAPK). Arrows point at calibration markers: ferritin 440 kDa; aldolase 156 kDa; albumin 67 kDa. The bulk of GDI is associated with Rab proteins and is thus present as complexes of ≈80 kD , which elute in fractions 8-9 on the Superose 12 column. (B) The Superose 12 fractions (A) were pooled 2 by 2 and assayed for their capacity to stimulate 1 µM GST-GDI to extract early endosomal Rab5 . After endosome removal on gradients, GST-GDI with bound Rab5 was retrieved onto glutathione beads and analyzed by western blotting. For comparison, the activity of 100 µg RLC or P30 is shown. The fractionation (A) and analysis (B) was repeated 3 times, and representative examples are shown. (C) HeLa cells were co-transfected with Flag-TXNL1 and His6-p38MAPK. Immunoprecipitation (IP) from lysates (lys: 5% of total) was with (+) or without (−) the indicated antibodies, and analysis was by western blotting. Each experiment was repeated at least 3 times, and (C) shows a representative example. (D) The outline of TXNL1 is shown with the CPGC motif in the TRX-like N-terminal domain (residues 1–107). Purified recombinant proteins were assayed (5 µM) before or after GST cleavage for their capacity to reduce insulin disulfide bonds in the presence of dithiothreitol, using dithiothreitol alone as a control (CTRL). Data are normalized to values obtained with E.Coli thioredoxin (TRX).
Figure 2
Figure 2. Association with early endosomes.
(A) Early endosomes were prepared from BHK cells treated or not for 10 min with 0.05 or 1 mM H2O2 , and analyzed by western blotting. (B) Experiments were as in (A), but cells were transfected with Flag-TXNL1 or Flag-TXNL1C34,37S. Open and solid arrows point at Flag-tagged and endogenous TXNL1, respectively. Each experiment in (C) and (D) was repeated at least 5 times, and representative examples are shown. (C) Phospho-p38MAPK present in early endosomal fractions was quantified after lysis in 1% TX100 by sandwich ELISA (expressed as OD units per mg protein). The figure shows the mean of 3 experiments. Error bars represent standard deviations; the observed differences are significant according to Student's t test with p<0.0001 at each H2O2 concentration. (D) HeLa cells were analyzed by immunofluorescence using antibodies against phospho-p38MAPK (pp38) and Rab5, followed by labeled secondary antibodies. Arrows point at examples of endosomes containing both Rab5 and pp38 (see inset). A fraction of pp38MAPK colocalized with Rab5 on early endosomes, but we were unable to unambiguously localize pp38MAPK on the membrane of other organelles using various markers, presumably because detection was limiting. (E) Cells were processed as in (D), except that primary antibodies against phospho-p38MAPK were omitted, and then analyzed in the red channel.
Figure 3
Figure 3. Rab5 capture by GDI is modulated by TXNL1.
(A) The capture of early endosomal Rab5 by GDI was analyzed. In the assay, GST-GDI was incubated with early endosomes in the absence (−) or presence of cytosol prepared from mock-transfected cells (mock), or from cells overexpressing either the Rab5 effector Rabankyrin-5 (RabAnk-5) or TXNL1. Rab5 bound to GDI was then retrieved and analyzed as in Fig 1B. The experiment was repeated 3 times, and a representative example is shown. (B) The capture of early endosomal Rab5 by GDI was analyzed as in (A), except that the assay was in the absence (−) or presence (+) of rat liver cytosol and 500 nM recombinant TXNL1 or TXNL1C34, 37S (after GST removal). Recombinant proteins were pre-reduced for 30 min with 1 mM DTT on ice (reducing conditions), or pre-oxidized for 15 min with 10 mM H2O2 at 25°C (oxidizing conditions). In both cases, proteins were then diluted 12 fold in the reaction mixture and the same final DTT or H2O2 concentrations were used when TXNL1 or TXNL1C34, 37 were omitted. These experiments were repeated 5 times, and representative examples are shown (C) Pulldown of rat liver cytosol (RLC) using purified GST fusion proteins that were (+) or not (−) pre-oxidized (Ox) as in (B). GST-proteins were retrieved onto immobilized beads and analyzed by western blotting with anti-Rab5 antibodies (all lanes are from the same gel). These experiments were repeated 4 times, and representative examples are shown. All lanes in the panel are from the same experiment. (D) Pulldown of rat liver cytosol (RLC) using purified GST-fusion proteins that were (+) or not (−) pre-reduced (Red) or pre-oxidized (Ox) as in (B). Analysis was by western blotting with anti-GST or anti-GDI antibodies. These experiments were repeated 3 times, and representative examples are shown (E) Pulldowns with GST-fusion proteins (C, and see Fig 1D) were analyzed by western blotting with anti-GDI antibodies. These experiments were repeated 3 times, and representative examples are shown (F) HeLa cells were either mock transfected or transfected with siRNA duplexes for 72 h (MGC2477 hypothetical protein as RNAi control). Total cell-extracts were analyzed by western blotting with the indicated antibodies.
Figure 4
Figure 4. TXNL1 regulates fluid phase endocytosis.
(A) HRP uptake for 5 min at 37°C was quantified in HeLa cells either mock-transfected or transfected with TXNL1-siRNA duplexes. Data are normalized to the uptake in mock-treated cells and are representative of at least three independent experiments (each in duplicate). The error bar represents the standard deviation; the observed differences are significant according to Student's t test with p<0.005 (B) HeLa cells were either mock transfected or transfected with Flag-TXNL1 or Flag-TXNL1C34, 37S. Cells were incubated with HRP for 5 min at 37°C and analyzed as in (A). The figure shows the mean of 3 independent experiments. Error bars represent the standard deviation; the observed differences are significant according to Student's t test with p<0.0001 under each condition (C–D) HeLa cells were either mock transfected or transfected with Flag-TXNL1 or Flag-TXNL1C34, 37S, as in (B), incubated with rhodamine-labeled transferrin (Tf), processed for immunofluorescence using anti-flag antibodies and visualized by double-channel fluorescence microscopy (D). The number of transferrin-containing endosomes was counted in 20 cells of duplicate samples in 3 independent experiments (as in D) and the quantification is shown in (C). Error bars represent the standard deviation; the observed differences after TXNL1C34, 37S overexpression are significant (p<0.01), but not after TXNL1 overexpression (p<0.2).
Figure 5
Figure 5. The TXNL1 cycle.
The model outlines the proposed mechanism by which TXNL1 may regulate fluid phase endocytosis, by modulating GDI function in the membrane-cytosol cycle of Rab5. See text for further explanations. P38, p38MAPK; pP38, phosphorylated p38MAPK; GDI, Guanine nucleotide Dissociation Inhibitor; pGDI; phosphorylated GDI; Rab5∶GTP or GDP, Rab5 in the GTP- or GDP-bound state, respectively.
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