BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity - PubMed (original) (raw)

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Hye-Won Shin et al. Mol Biol Cell. 2004 Dec.

Abstract

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.

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Figures

Figure 1.

Figure 1.

Localization of BIG2. HeLa cells expressing HA-tagged BIG2(WT) were fixed and double stained for HA and either the AP-1 γ-adaptin subunit (A), GGA3 (B), CI-MPR (C), EEA1 (D), or TfnR (E) followed by Cy3-conjugated anti-rat IgG and Alexa488-conjugated anti-mouse IgG.

Figure 2.

Figure 2.

Effects of Rab11(S25N) expression and BFA treatment. (A) HeLa cells expressing HA-tagged BIG2(WT) and FLAG-tagged Rab11(S25N) were serum starved and allowed to internalize Alexa488-conjugated Tfn for 60 min at 37°C as described under Materials and Methods. The cells were fixed and stained with rat monoclonal anti-HA and mouse monoclonal anti-FLAG antibodies followed by Cy3-conjugated anti-rat and Cy5-conjugated anti-mouse secondary antibodies. (B) HeLa cells expressing HA-tagged BIG2(WT) were treated with vehicle only (top) or with 5 μg/ml BFA (bottom) for 15 min and then fixed and double stained for HA and TGN46 followed by Cy3-conjugated anti-rat and Alexa488-conjugated anti-rabbit secondary antibodies. Bar, 10 μm.

Figure 3.

Figure 3.

Immunofluorescence analysis of cells expressing BIG2(E738K). HeLa cells expressing HA-tagged BIG2(E738K) were fixed and double stained for HA and either the AP-1 γ-adaptin subunit (A and B), TGN46 (C), TfnR (D), or EEA1 (E). Bar, 10 μm.

Figure 4.

Figure 4.

Localization of Rab4 and Rab11, but not Rab5 or Rab7, to the BIG2(E738K)-induced tubular structures. HeLa cells expressing HA-tagged BIG2(E738K) and either EGFP-tagged Rab5 (A) or Rab4 (B), or ECFP-tagged Rab11 (C) or Rab7 (D) were fixed and stained with anti-HA antibody. Bar, 10 μm.

Figure 5.

Figure 5.

Incorporation of internalized Tfn and Stx1 but not EGF into the BIG2(E738K)-induced tubular structures. (A) HeLa cells expressing HA-BIG2(E738K) were serum starved for 3 h and incubated with Alexa488-conjugated Tfn at 37°C for 2.5, 5, or 30 min. (B) HeLa cells expressing HA-BIG2(E738K) were incubated with Cy3-conjugated Stx1 on ice for 50 min, washed, and incubated at 37°C for 5, 20, or 60 min. (C) HeLa cells expressing HA-BIG2(E738K) were serum starved for 3 h and incubated with Alexa488-conjugated EGF at 37°C for 5, 10, or 30 min. The cells were then fixed and stained with anti-HA antibody followed by Cy3-conjugated (A and C) or Alexa488-conjugated (B) anti-rat secondary antibodies. Bar, 10 μm.

Figure 6.

Figure 6.

Tfn recycling is not affected by the BIG2 (E738K) expression. HeLa cells expressing HA-tagged BIG2(WT) (A) or BIG2(E738K) (B) were serum starved for 3 h and incubated with Alexa488-conjugated Tfn at 37°C for 60 min. The cells were washed extensively and incubated with medium that did not contain labeled Tfn at 37°C for indicated periods of time. Bar, 10 μm.

Figure 7.

Figure 7.

Internalized TGN38 enters the BIG2(E738K)-induced tubular structures. HeLa cells coexpressing FLAG-TGN38 and either HA-tagged BIG2(WT) (A) or BIG2(E738K) (B) were incubated with anti-FLAG M2 antibody on ice for 50 min, washed, and further incubated at 37°C for 10, 30, or 60 min. Bar, 10 μm.

Figure 8.

Figure 8.

BIG2 has a GEF activity toward ARF1 and ARF3 in vivo. (A) HeLa cells were transfected with the indicated expression vectors and then lysed. The cell lysates (∼500 μg of protein) were subjected to pull-down assay with the GST-GGA1-GAT domain as described under Materials and Methods (bottom). Top and middle, expression levels of HA-BIG2 and ARF-HA, respectively, by subjecting the cell lysates directly to Western blot analysis by using anti-HA antibody. (B) Fold activation of each ARF by coexpressed BIG2. The ratios of bound versus total ARFs were estimated from the band intensities in the middle and bottom panels in A. This is the representative of three independent experiments.

Figure 9.

Figure 9.

Dominant-negative mutants of ARF1 and ARF3 enhance the BIG2(E738K)-induced tabulation. HeLa cells coexpressing untagged BIG2(E738K) and either HA-tagged ARF1(T31N) (A), ARF3(T31N) (B), ARF5(T31N) (C), or ARF6(T27N) (D) were fixed and double stained with polyclonal rabbit anti-BIG2 and monoclonal rat anti-HA antibodies followed by Cy3-conjugated anti-rabbit and Alexa488-conjugated anti-rat secondary antibodies. In C, red arrows indicate the shortened tubules. Bar, 10 μm.

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