MARCH-II is a syntaxin-6-binding protein involved in endosomal trafficking

Abstract

Membrane-associated RING-CH (MARCH) is a recently identified member of the mammalian E3 ubiquitin ligase family, some members of which down-regulate the expression of immune recognition molecules. Here, we have identified MARCH-II, which is ubiquitously expressed and localized to endosomal vesicles and the plasma membrane. Immunoprecipitation and in vitro binding studies established that MARCH-II directly associates with syntaxin 6. Overexpression of MARCH-II resulted in redistribution of syntaxin 6 as well as some syntaxin-6-interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into the MARCH-II-positive vesicles. In addition, the retrograde transport of TGN38 and a chimeric version of furin to trans-Golgi network (TGN) was perturbed--without affecting the endocytic degradative and biosynthetic secretory pathways--similar to effects caused by a syntaxin 6 mutant lacking the transmembrane domain. MARCH-II overexpression markedly reduced the cell surface expression of transferrin (Tf) receptor and Tf uptake and interfered with delivery of internalized Tf to perinuclear recycling endosomes. Depletion of MARCH-II by small interfering RNA perturbed the TGN localization of syntaxin 6 and TGN38/46. MARCH-II is thus likely a regulator of trafficking between the TGN and endosomes, which is a novel function for the MARCH family.

Figure 5.

Redistribution of syntaxin-6 partners. Right two panels, COS7 cells transfected with MARCH-II (A), FLAG-VAMP3 plus GFP-MAR2 (B), FLAG-VAMP4 plus GFP-MAR2 (C), or GFP-MAR2 (D). Left, control cells transfected with mock (A and D), FLAG-VAMP3 (B), or FLAG-VAMP4 (C). After 18 h of transfection, cells were processed for immunofluorescence analysis with antibodies for MARCH-II, Vti1a, FLAG-tag, and syntaxin 8 followed by Alexa 488-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG. The signals for SNAREs are shown in red and those for MARCH-II/GFP-MAR2 are shown in green. Arrowheads indicate the colocalization of two proteins. Asterisks, the position of nucleus. Bars, 10 μm.

Figure 6.

Overexpression of MARCH-II perturbs the localization and transport of TGN38 and furin. (A) COS7 cells expressing TGN38 alone (a) or together with GFP-MAR2 (b and b′) were fixed, permeabilized, and stained with anti-TGN38 followed by TRITC-conjugated secondary antibody. (B) Cells expressing TGN38 alone (c) or together with Myc-syn6cyto (d) or with GFP-MAR2 (e and e′) were incubated with anti-TGN38 for 40 min and subsequently chased for 1 h at 37°C. Endocytosed antibodies were visualized with TRITC-conjugated secondary antibody. (C) Cells expressing FLAG-furin alone (f) or together with GFP-MAR2 (g and g′) were fixed, permeabilized, and stained with anti-FLAG followed by TRITC-conjugated secondary antibody. (D) Cells expressing FLAG-furin alone (h) or together with Myc-syn6cyto (i) or with GFP-MAR2 (j and j′) were incubated with anti-FLAG for 40 min and subsequently chased for 1 h at 37°C. Endocytosed antibodies were visualized with TRITC-conjugated secondary antibody. Arrowheads in (A–D) indicate the colocalization of two proteins. (E) Cells coexpressing furin, MPR-FLAG, and GFP-MAR2 were stained with anti-furin and anti-FLAG followed by Alexa 350- and TRITC-conjugated secondary antibodies, respectively. (F) Cells coexpressing furin and GFP-MAR2 were stained with anti-furin and anti-EEA1 followed by Alexa 350- and TRITC-conjugated secondary antibodies, respectively. Arrowheads in E and F indicate the positions of the vesicles positive for both GFP-MAR2 and furin. Asterisks, the position of nucleus. Bars, 10 μm (A–D) or 5 μm (E and F).

Figure 7.

MARCH-II overexpression affects Tf uptake and distribution of TfR. (A and G) COS7 cells transfected with MARCH-II were double stained with anti-MAR2_C#41 and anti-TfR, and the signals were observed with confocal microscopy. Images in G were focused on the cell surface area. (B) COS7 cells transfected with GFP-MAR2 were stained with anti-Rab11. (C–E) COS7 cells transfected with either FLAG-Rab11 (C), FLAG-Rab11 plus GFP-MAR2 (D), or MARCH-II (E) were incubated with TMR-Tf at 37°C for 1 h. Cells were washed, fixed, and stained with anti-FLAG (C and D) or with anti-MAR2_C#41 and anti-TfR (E). (F) COS7 cells expressing GFP-MAR2 were incubated with TMR-Tf at 37°C for 5 min (top) or 30 min (bottom). Cells were washed, fixed, and observed by fluorescence microscopy. Asterisks indicate cells expressing GFP-MAR2. Bars, 10 μm (A–G). (H) HeLa cells transfected with an MARCH-II (open circles) or an empty vector (closed circles) were incubated with ¹²⁵I-Tf at 37°C for 5, 15, 30, and 60 min. After washing with acidic buffer to release the cell surface-associated ¹²⁵I-Tf, the amounts of the internalized radioactivity were determined. Data are represented as the mean from three independent experiments. (I) HeLa cells were transfected with GFP (lanes 1 and 2) or GFP-MAR2 (lane 3). At 18 h after transfection, cells were incubated in HBSS (lane 1) or HBSS containing 0.5 mg/ml EZ-Link Sulfo-N-hydroxysuccinimide-LC-Biotin (lanes 2 and 3) on ice for 30 min. After washing with ice-cold 0.1 M Tris-HCl, pH 7.5, cells were extracted in radioimmunoprecipitation assay lysis buffer. Lysate (300 μg of proteins) was incubated with streptavidin–agarose beads and the bound materials (biotinylated cell surface proteins) were eluted by boiling in Laemmli sample buffer. Whole cell lysates (10 μg of proteins; top) and biotinylated samples (bottom) were subjected to immunoblot analysis with anti-TfR. Asterisk indicates dimeric forms of TfR.

Figure 8.

EGF trafficking and EGFR down-regulation are not affected by MARCH-II overexpression. (A–D) COS7 cells expressing GFP-MAR2 were incubated with TMR-epidermal growth factor for 5 min (A), 10 min (B), or 30 min (C) at 37°C. After a 30-min loading, cells were washed and incubated in marker-free medium for 2 h at 37°C (D). Merged images of fluorescence signals for GFP (green) and TMR (red) are shown. Overlaps of both signals are indicated by arrows. Bars, 20 μm. (E) HeLa cells transfected with MARCH-II (right) or an empty vector (left) were starved by a 1-h incubation in Opti-MEM I at 37°C. Cells were subsequently treated with 1 μg/ml EGF for indicated time at 37°C. Each sample (8 μg of protein) was subjected to immunoblot analysis with anti-EGFR (top), anti-calnexin (middle), and anti-MAR2_C#41 (bottom).

Figure 9.

Specific effects of MARCH-II suppression on syntaxin 6 and TGN38/46. (A) Northern blot of HeLa-TGN38 cells (control) or those stably expressing the MARCH-II shRNA (siRNA) (20 μg of total RNA each) was hybridized with a specific probe for human MARCH-II (top). The blot was subsequently reprobed with BiP/GRP78 cDNA as a loading control (bottom). (B) Control or the MARCH-II shRNA expressing HeLa-TGN38 cells were transiently transfected with human MARCH-II and lysed in radioimmunoprecipitation assay buffer at 18 h after transfection. Whole cell lysates (10 μg of each protein) were subjected to immunoblot analysis with indicated antibodies. (C) HeLa-TGN38 cells (right) and those ex pressing the MARCH-II shRNA (left) were incubated with anti-TGN38 for 40 min and subsequently chased in presence of Alexa 488-Tf for 1 h at 37°C. Endocytosed antibodies were visualized with Alexa 546-conjugated secondary antibody. Right and left, signal for anti-TGN38. Middle, magnified image of inset in left image, which is merged with signals for anti-TGN38 (red) and Alexa 488-Tf (green). Arrowheads indicate colocalization of both signals. (D) HeLa-TGN38 cells (right) and those expressing the MARCH-II shRNA (left) were incubated with Alexa 488-Tf for 1 h at 37°C and subsequently stained with anti-TGN46 followed by Alexa 546-conjugated secondary antibody. Middle, magnified image of inset in left, which is merged with signals for anti-TGN46 (red) and Alexa 488-Tf (green). Arrowheads indicate colocalization of both signals. (E) HeLa-TGN38 cells expressing the MARCH-II shRNA were transiently transfected with FLAG-furin. Cells were incubated with anti-FLAG for 40 min and subsequently chased for 1 h at 37°C. Endocytosed antibodies were visualized with Alexa 546-conjugated secondary antibody. (F and G) HeLa-TGN38 cells expressing the MARCH-II shRNA were labeled by 1-h incubation with Alexa 488-Tf (F) or TMR-epidermal growth factor (G) at 37°C, and subsequently washed and fixed. Nucleus (blue) was labeled with Hoechst 33342. (H and I) HeLa-TGN38 cells (right) and those expressing the MARCH-II shRNA (left) were stained with anti-syntaxin 6 (H) or anti-β1,4-galactosyltransferase (I) followed by Alexa 546-conjugated secondary antibody. Bars, 5 μm (C and D) or 10 μm (E–I).

Figure 10.

Characterization of the PDZ-binding motif of MARCH-II. (A) GST pull-down assay. Glutathione beads containing GST or GST-Veli were incubated with either His-MAR2_C or His-MAR2_C-244K. Resin-bound proteins (1/15 of the eluates) were separated by SDS-PAGE and visualized by silver staining. One-tenth of the amount of His-MAR2_C or His-MAR2_C-244K used in the binding reaction was run in parallel (input). (B–D) COS7 cells transfected with GFP-MAR2–244K were processed for immunofluorescence analysis with an antibody against calreticulin (B), syntaxin 6 (C), or TGN46 (D) followed by Alexa 546-conjugated secondary antibody. (E) COS7 cells transfected with GFP-MAR2–244K were labeled with TMR-Tf for 30 min at 37°C. Cells were subsequently washed, fixed, and observed by fluorescence microscopy. The signals for GFP are shown as green and those for the marker proteins are shown as red. Bars, 10 μm.

Figure 1.

Primary structure, tissue distribution, and subcellular fractionation of rat MARCH-II. (A) The predicted amino acid sequence of rat MARCH-II is shown. A PHD finger motif (shadowed), hydrophobic regions (underlines), and a PDZ-domain binding motif (a wavy line) are indicated. (B) Rat multiple Northern blots (2 μg of polyadenylated RNA each) were hybridized with the specific probe for MARCH-II (top). The blots were subsequently reprobed with β-actin cDNA (bottom). (C) HeLa cells transfected with rat MARCH-II were homogenized in HBSS and ultracentrifuged. The membrane pellets were incubated with HBBS, 2 M NaCl, 0.15 M NaHCO₃, pH 11.5, or 1% Triton X-100 for 1 h at 4°C. After high-speed centrifugation, the supernatant (S) and pellets (P) were analyzed by immunoblotting with anti-MAR2_C#41 (top) or anti-calnexin (bottom). (D) The homogenate of rat liver was subjected to discontinuous sucrose density gradient centrifugations as described in Materials and Methods, yielding membrane fractions enriched in the PMs, LMs, HMs, and Ls. Each fraction (10 μg of protein) was subjected to immunoblotting with anti-MAR2_C#41 and antibodies against the indicated proteins.

Figure 2.

Subcellular localization of endogenous MARCH-II in CHO cells. (A) CHO cells were fixed, permeabilized, and stained with anti-MAR2_N#51 antiserum followed by Alexa 488-conjugated anti-rabbit IgG. Punctate staining was observed within the cytoplasm. Occasionally, intense staining was detected at the edge of the cell (arrows). Asterisks, position of nucleus. Bar, 10 μm. (B) CHO cells were processed for double-labeled confocal microscopy with anti-MAR2_N#51 and a mouse monoclonal antibody against syntaxin 6 (a–a″), EEA1 (b–b″), γ-adaptin (c–c″), or TfR (d–d″). Primary antibodies were visualized with Alexa 546-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG. The signals for endogenous MARCH-II are shown in green, and those for the endogenous marker proteins are shown in red. Arrowheads indicate the colocalization of MARCH-II with syntaxin 6. Asterisks, the position of nucleus. Bars, 10 μm. (C) Higher magnified images of insets in B are shown. Arrows indicate examples of colocalization. Bars, 1 μm.

Figure 3.

MARCH-II interacts with syntaxin 6. (A) Triton extracts of rat liver Golgi-rich membranes were incubated with either Talon resins alone (lane 2) or the resins immobilized with His-MAR2_C (lane 3). Proteins bound to the resins were eluted and subjected to immunoblotting with antibodies against the indicated proteins. One-eightieth of the amount used in the binding reaction was run in parallel (lane 1). (B) Extracts from COS7 cells expressing MARCH-II alone (lane 1) or together with FLAG-Syn6 (lane 2) were incubated with anti-FLAG M2 agarose. After extensive washing, the eluates were subjected to immunoblotting with anti-FLAG M2 (top) or anti-MAR2_N#384 (middle). Extracts (2% of input) were immunoblotted with anti-MAR2_N#384 to confirm expression of MARCH-II (bottom). (C) Schematic representation of GST-fusion proteins of syntaxin 6 and VAMP3 used in GST pull-down assay. (D) Glutathione beads containing GST or GST-fusion proteins were incubated with His-MAR2_C. Resin-bound proteins (one-third of the eluate) were separated by SDS-PAGE and visualized by silver staining for GST or GST-fusion proteins (top) or by immunoblotting with anti-MAR2_C#41 for His-MAR2_C (bottom). One-tenth of the amount of His-MAR2_C used in the binding reaction was run in parallel (lane 1).

Figure 4.

Recruitment of syntaxin 6 to the MARCH-II vesicles. (A–C) COS7 cells transfected with MARCH-II were double-stained with anti-MAR2_C#41 (1:1000) and a mouse monoclonal antibody against syntaxin 6 (A), EEA1 (B), or γ-adaptin (C) followed by Alexa 546-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG. (D) COS7 cells transfected with GFP-MAR2 were stained with anti-β1,4-galactosyltransferase followed by Alexa 546-conjugated anti-goat IgG. The signals were observed by confocal microscopy and those for MARCH-II/GFP-MAR2 and the marker proteins are shown as green and red, respectively. In contrast to the perinuclear localization in untransfected cells (triangles in A, merge), redistribution of syntaxin 6 to the MARCH-II endosomes is observed in cells overexpressing MARCH-II (asterisk). Asterisks and triangles, the position of nucleus. Bars, 10 μm.