α-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

<div><p>Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.</p></div

Knockdown of α-taxilin induces the degradation of TfnR.

<p>(A) Top: HeLaS3 cells transfected with control (Con) or α-taxilin siRNA (#2, #3 and #4) were lysed, and the cell lysates were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.0005; **, P<0.005; ***, P<0.005, by Student's <i>t</i>-test. (B) Top: HeLaS3 cells stably expressing control (Con) or α-taxilin shRNA (#2, #7) were lysed, and the cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.005; **, P<0.005, by Student's <i>t</i>-test. (C) Total RNA was extracted from HeLaS3 cells transfected with control (Con) or α-taxilin siRNA (#2, #3 and #4) for 48 h, and <i>TFNR</i> and <i>GAPDH</i> mRNA were analyzed by RT-PCR. The ratio of the <i>TFNR</i> mRNA level relative to the <i>GAPDH</i> mRNA level was expressed as arbitrary units. <i>TFNR</i> mRNA level relative to <i>GAPDH</i> mRNA level in control HeLaS3 cells was set to 1.0. The results shown are means ± s.e.m. from three independent experiments. Ns, not significant, by Student's <i>t</i>-test. (D) Top: HeLaS3 cells transfected with control (Con) or α-taxilin (αTax) siRNA (#3) were treated with 0.1% DMSO, 10 μM lactacystin or 100 nM bafilomycin A1 (BafA1) for 24 h. The cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.005; **, P<0.01; ns, not significant, by Student's <i>t</i>-test. (E) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with 0.1% DMSO or 100 nM bafilomycin A1 (BafA1) for 24 h, and then the cells were immunostained with anti-TfnR and anti-α-taxilin antibodies. The results shown are representative of three independent experiments. Scale bars, 10 μm. (F) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were stimulated with EGF (100 ng/ml) for the indicated time periods. Cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. (G) The amount of EGFR in (F) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of EGFR at each time point to EGFR at time zero from three independent experiments. Values at time zero are set to 100%. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 15, 30, 60 min) determined by Student's <i>t</i>-test was not significant.</p

Knockdown of α-taxilin impedes the recycling of Tfn.

<p>(A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's <i>t</i>-test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's <i>t</i>-test. *, P<0.005; **, P<0.001. <i>P</i>-values at 10 min was not significant. (E) The intensity of Tfn-488 signal of HeLaS3 cells treated or untreated with 200 μg/ml leupeptin in (C) was calculated as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at 20 and 40 min to Tfn-488 at time zero. Values at time zero are set to 1.0. <i>P</i>-values (the cells untreated with leupeptin vs. the cells treated with leupeptin at 20 and 40 min) are determined by Student's <i>t</i>-test. *, P<0.05; **, P<0.05; ns, not significant. Con, control; α-Tax, α-taxilin.</p

α-Taxilin does not localize to endosome structures.

<p>(A) Total RNA was extracted from HeLaS3 cells transfected with control or SNX4 siRNA (#1, #2) for 48 h, and <i>SNX4</i> and <i>GAPDH</i> mRNA were analyzed by RT-PCR. The ratio of the <i>SNX4</i> mRNA level relative to the <i>GAPDH</i> mRNA level was expressed as arbitrary units. <i>SNX4</i> mRNA level relative to <i>GAPDH</i> mRNA level in control HeLaS3 cells was set to 1.0. The results shown are means ± s.e.m. from three independent experiments. *, P<0.005; **, P<0.005, by Student's <i>t</i>-test. (B) HeLaS3 cells transfected with control or SNX4 siRNA (#1, #2) were homogenized, and the post-nuclear supernatant (P) of the homogenate was separated into the cytosol (C) and membrane (M) fractions. Fractions were probed with the indicated antibodies. Syntaxin 4 and α-tubulin were used as markers for membrane and cytosol protein, respectively. (C) Top: HeLaS3 cells were immunostained with anti-EEA1 and anti-α-taxilin antibodies. Middle: HeLaS3 cells transfected with HA-Rab11 were immunostained with anti-HA and anti-α-taxilin antibodies. Bottom: HeLaS3 cells transfected with GFP-Rab7 were immunostained with an anti-α-taxilin antibody. Boxed areas are shown at higher magnification in the inset. Scale bars, 10 μm. The results shown are representative of three independent experiments.</p

New insights into the dimerization of small GTPase Rac/ROP guanine nucleotide exchange factors in rice

<div><p>Molecular links between receptor-kinases and Rac/ROP family small GTPases mediated by activator guanine nucleotide exchange factors (GEFs) govern diverse biological processes. However, it is unclear how the Rac/ROP GTPases orchestrate such a wide variety of activities. Here, we show that rice OsRacGEF1 forms homodimers, and heterodimers with OsRacGEF2, at the plasma membrane (PM) and the endoplasmic reticulum (ER). OsRacGEF2 does not bind directly to the receptor-like kinase (RLK) OsCERK1, but forms a complex with OsCERK1 through OsRacGEF1 at the ER. This complex is transported from ER to the PM and there associates with OsRac1, resulting in the formation of a stable immune complex. Such RLK-GEF heterodimer complexes may explain the diversity of Rac/ROP family GTPase signalings.</p></div

Activity of myosin phosphatase is bistable.

<p>[MP] denotes the concentration of activated myosin phosphatase. The concentration of activated myosin phosphatase is assumed to represent that of non-phosphorylated MYPT1. d[MP]/dt indicates the differentiation of [MP] against time t. (A) The plots are d[MP]/dt against [MP]. In the steady states, the reaction velocity is zero, whereby the steady states are d[MP]/dt = 0. Three intersections with the horizontal axis represent three steady states (S1, S2, and S3). (B) d[MP]/dt was plotted against [MP] using the various kcat₂ values. (C) d[MP]/dt was plotted against [MP] using the various kcat₁ values.</p

Mathematical modeling and simulation of MLC phosphorylation regulated by Rho-kinase.

<p>(A) Schematic overview of MLC phosphorylation regulated by Rho-kinase signal. Arrows, dashed line and - indicate stimulatory enzymatic reactions, myosin phosphatase auto-dephosphorylation signaling pathway and phosphorylation, respectively. (B) The graph shows that the transient Rho-kinase activations (red and blue line) are given as the input signal in our simulation. (C)(D) The simulation results of MLC and MYPT1 phosphorylation induced by the transient Rho-kinase activation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039269#pone-0039269-g001" target="_blank">Figure 1B</a>. (E)(F) The phosphorylation of MYPT1 and MLC against time t was calculated using the various kcat₂ values. (G) The phosphorylation of MLC against time t was calculated using the different kcat₃ values.</p

The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

<div><p>The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its <i>Arabidopsis</i> ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 <i>in vitro</i> and <i>in vivo</i>. SPL11 ubiquitinates SPIN6 <i>in vitro</i> and degrades SPIN6 <i>in vivo</i> via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of <i>Spin6</i> in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen <i>Magnaporthe oryzae</i> and the bacterial blight pathogen <i>Xanthomonas oryzae</i> pv. <i>oryzae</i>. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the <i>Spin6</i> RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state <i>in vitro</i> and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.</p></div

SPIN6 is a Rho GTPase-activating protein (RhoGAP) and interacts with SPL11 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The protein structure of SPIN6, SPIN6.2 and SPIN6.3. The position of the Pleckstrin Homology (PH) domain (34–142), RhoGAP domain (192–383), and coiled coil (CC) motif (583–699) are indicated. (B) SPIN6 interacts with SPL11 in yeast. SPL11 represents the full-length SPL11; ARM is the ARM domain of SPL11; and SPL11m is the SPL11 mutant with a three amino-acid deletion at C314P315T316 in the U-box domain, resulting in a loss-of-function of E3 ligase activity (Zeng et al., 2004). After they were diluted 10, 100, and 1000 times with sterilized-distilled H₂O, the Mav203 yeast transformants were plated on synthetic dextrose medium without Trp, Leu, and His amino acids (SD-LTH) and with 0 mM or 40 mM 3-amino-1,2,4,-triazole (3AT), separately. (C) SPIN6 binds to SPL11 in the GST pull-down assay. His:SPIN6 and GST:SPL11, GST:ARM of SPL11 were used in the assay. (D) SPIN6 interacts with SPL11 in <i>N</i>. <i>benthamiana</i> in BiFC assay. SPIN6 was fused with the C-terminal of eYFP to make SPIN6:CeYFP. SPL11, ARM, and SPL11m were fused with the N-terminal eYFP to make NeYFP:SPL11, NeYFP:ARM, and NeYFP:SPL11m, respectively.</p

Proposed working model of the relationship between SPL11, SPIN6, and OsRac1.

<p>By associating with RhoGAP protein SPIN6, the E3 ligase SPL11 negatively modulates OsRac1-mediated immune signaling. SPIN6 is ubiquitinated and degraded by the E3 ligase SPL11 via the 26S proteasome system. From the GDP state to the GTP state, OsRac1 is activated by the GEF protein OsRacGEF1, then associates with the NADPH oxidases OsRBOH/CDPK complex to trigger ROS generation. The activation of OsRac1 requires the phosphorylation by the kinase protein OsCERK1, a co-receptor of the PAMP effector chitin. OsCERK1 dimerizes with LysM protein CEBiP1 to perceive chitin signaling. The interaction between SPIN6 and OsRac1 may lead to the change of OsRac1 from the GTP state to the GDP state, which reduces the active form of OsRac1 in rice cells. Mutation in the <i>Spin6</i> gene may cause accumulation of ROS and PR proteins, i.e., PR1a, PR5 and PBZ1, that results in plant cell death and immunity.</p