Renigunta V et al. (2014), Cooperative endocytosis of the endosomal SNARE ...

XB-ART-48911

Mol Biol Cell 2014 Jun 15;2512:1877-91. doi: 10.1091/mbc.E13-10-0592.

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Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

Renigunta V , Fischer T , Zuzarte M , Kling S , Zou X , Siebert K , Limberg MM , Rinné S , Decher N , Schlichthörl G , Daut J .

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The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

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Species referenced: Xenopus laevis
Genes referenced: cltc kcnk3 kcnk9 pigy rab5a stx7 stx8 vamp8 vti1b

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	FIGURE 1:. The K2P channel TASK-1 interacts with the SNARE protein syntaxin-8. (A) The topology of TASK-1 and stx8. (B) Membrane yeast two-hybrid screen with TASK-1 or TASK-3 as bait and stx8 or mutants thereof as prey. The Q179A mutant of stx8 cannot assemble with other SNARE proteins; in the Δ100–140 mutant the linker between the Hc domain and the SNARE domain was excised. (C) RT-PCR analysis of TASK-1 and endosomal SNARE proteins in human brain and in A549 cells. Asterisks represent nonspecific PCR products. (D) Coimmunoprecipitation of stx8 and TASK-1 endogenously expressed in A549 cells. The complex containing TASK-1 was precipitated from cell lysate with a TASK-1–specific antibody from Alomone (APC-024), and a Western blot of the precipitate was probed with TASK-1, stx8, and stx7 antibodies (left); the cell lysate (input) was used as positive control. Coimmunoprecipitation with an unrelated immunoglobulin G antibody (Santa Cruz Biotechnology) was used as a negative control (right).
	FIGURE 2:. Coexpression of TASK-1 with stx8 or stx7 in CHO cells and Xenopus oocytes. (A) Current–voltage relation of rTASK-1 expressed in CHO cells. The currents were measured using voltage ramps from −120 to +40 mV at pH 7.4 (black curve) and 6.0 (red curve). (B) TASK-1 current–voltage relation measured in the same batches of CHO cells 48 h after transfection of TASK-1 alone (black curve) and after cotransfection of TASK-1 with stx8 (green curve); mean values ± SEM of n = 28 cells. (C) Mean outward currents ± SEM measured in CHO cells at 0 mV after transfection with rat TASK‑1 alone (black) and after cotransfection of TASK-1 with stx8 (green) or stx7 (orange). (D) Typical current–voltage relation measured 48 h after injection of human TASK-1 cRNA (black curve) and after coinjection of TASK-1 and stx8 cRNA. For experiments with human TASK-1 we used the NQTASK-1 mutant, which displays a higher current amplitude (Zuzarte et al., 2009; Materials and Methods). (E) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV after injection of hTASK‑1 or hTASK-3 cRNA alone (black) or together with 1.5, 3, or 6 ng stx8 cRNA per oocyte as indicated. (F) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV measured after injection of hTASK‑1 cRNA alone (black) or together with 6 ng cRNA encoding stx8, VAMP8, vti1b, or stx7. (G) Mean surface expression of HA-tagged hTASK-1 channels (measured in relative light units [RLUs]) in Xenopus oocytes after injection of TASK-1 cRNA alone or together with 6 ng of stx8 or stx8Q179A. (H) Mean hTASK-1 current measured in Xenopus oocytes after injection of hTASK-1 cRNA alone or together with stx8 or stx8Q179A. In all bar graphs the number of oocytes or CHO cells from which the data were obtained is indicated in brackets. Note that in the series of experiments shown in E, F, and H, coinjection of 6 ng of stx8 cRNA caused a reduction of TASK-1 current to values between 13 and 23% of control, illustrating that there was a certain degree of variability among different batches of oocytes. For this reason, TASK-1 (and other) currents with and without coinjection of a second cRNA were always compared in the same batch of oocytes (measured on the same day); normalized current amplitudes of at least three different batches are combined in the bar graphs.
	FIGURE 3:. Dissection of the interacting regions of stx8 and TASK-1. (A) Topology of stx8, stx7, and the stx8/stx7 chimeras. (B) TASK-1 currents measured in Xenopus oocytes expressing TASK-1 and stx8 or stx8/stx7 chimeras. (C) Normalized hTASK-1 currents measured in Xenopus oocytes expressing hTASK-1 and stx8 or deletion mutants of stx8. (D) Normalized currents measured in Xenopus oocytes expressing TASK-3/TASK-1 or TASK-1/TASK-3 chimeras alone or together with stx8 or stx8Q179A. (E) Schematic drawing of TASK-3/TASK-1 and TASK-1/TASK-3 chimeras.
	FIGURE 4:. Clathrin-mediated endocytosis of TASK-1. (A) The effects of stx8 on TASK-1 currents in Xenopus oocytes with and without coexpression of AP180C, a suppressor of clathrin-mediated endocytosis. (B) The effects of mutating the dileucine-based endocytosis signal in stx8, the tyrosine-based endocytosis signal in TASK-1, or both on the amplitude of TASK-1 currents. For each batch of oocytes the currents were normalized to the currents measured with TASK-1 (or the mutant TASK-1Y300A) alone. (C) The effect of mutating the two endocytosis signals on the surface expression of HA-tagged TASK‑1 in Xenopus oocytes. The surface expression was measured using an antibody-based luminometric assay (Materials and Methods).
	FIGURE 5:. Analysis of endocytosis of TASK-1 using an antibody uptake assay. (A) COS-7 cells expressing HA epitope–tagged TASK-1 (or TASK-1 mutants) and stx8 (or stx8 mutants) were incubated with an anti-HA antibody at 4°C to label the channels at the cell surface and then warmed to 37°C to initiate internalization. After incubation at 37°C for 30 min, the anti-HA–labeled channels that remained on the surface were detected with a secondary antibody labeled with Alexa Fluor 594 (red). Then the cells were permeabilized, and the internalized channels were detected with a different secondary antibody, labeled with Alexa Fluor 488 (green). The measurements were carried out 48 h after transfection of TASK-1, TASK-1 mutants, stx8, stx8 mutants, or stx7. Note that rTASK‑1Y317A corresponds to hTASK-1Y300A. (B) Statistical evaluation of the antibody uptake assay under different conditions. The ratio between the fluorescence of internalized channels (green) and channels at the cell surface (red) was calculated at 0 and 30 min after heating to 37°C; number of cells is indicated in brackets. Scale bars, 50 μm.
	FIGURE 6:. Live-cell imaging of HeLa cells cotransfected with mCherry-tagged stx8 and EGFP-tagged hTASK-1. (A–G) Cotransfection of TASK-1 and stx8; the Pearson coefficient was 0.89 ± 0.02 (n = 7 cotransfection experiments, 37 cells). Plain arrows indicate colocalization; crossed arrows indicate lack of colocalization. (D–F) Higher magnifications of the region indicated by the square in C. (H–N) Cotransfection of TASK-1 and stx8Q179A; the Pearson coefficient was 0.86 ± 0.02 (n = 4 cotransfection experiments, 33 cells). (O–Q) Cotransfection of TASK-1 with stx8Δ216-233; the Pearson coefficient was 0.41 ± 0.03 (n = 4 transfection experiments, 33 cells). (K–M) Higher magnifications of the regions indicated by the square in J. All images were taken 48 h after transfection. Scale bars, 5 μm (A–C, G–I), 1 μm (D–F, K–M). (G, N) Intensity profiles of F and M (green line, EGFP; red line, mCherry). For calculating the Pearson coefficient, the entire cell was selected as region of interest.
	FIGURE 7:. Live-cell imaging of HeLa cells cotransfected with fluorescence-labeled TASK-1 and the endosomal marker 2xFYVE or rab5. (A–G) Cotransfection of mCherry-tagged TASK-1 and EGFP-tagged 2xFYVE. Similar results were obtained in n = 3 transfections. Plain arrows indicate colocalization; crossed arrows indicate lack of colocalization. (H–N) Cotransfection of mCherry-tagged rab5 and EGFP-tagged TASK-1. (D–F, K–M) Higher magnifications of the regions indicated by the squares in C and J. (G, N) Intensity profiles of F and M (green line, EGFP; red line, mCherry/DsRed). All images were taken 48 h after transfection. Similar results were obtained in n = 3 transfections. Scale bars, 5 μm (A–C, H–J), 1 μm (D–F, K–M).
	FIGURE 8:. TIRF microscopy of TASK-1 and stx8. (A, B) HeLa cells transfected with EGFP-tagged hTASK-1 and mCherry-tagged stx8; scale bars, 5 μm. (C) Sequence of TIRF images taken from the cell shown in A; scale bars, 0.5 μm. The two bottom rows show the red and green channels (Materials and Methods). TASK-1 and stx8 are colocalized in diffraction-limited spots for at least 37 s. (D) Sequence of TIRF images taken from the cell shown in B; scale bars, 0.5 μm. The two bottom rows show the red and green channels (Materials and Methods). TASK-1 and stx8 are colocalized in diffraction-limited spots for at least 4.6 s.

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