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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02524

Journal of Cell Science 118, 4039-4048 (2005)
Published by The Company of Biologists 2005
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Roles of ARFRP1 (ADP-ribosylation factor-related protein 1) in post-Golgi membrane trafficking

Hye-Won Shin1,2,*, Hiromi Kobayashi2,*, Masashi Kitamura1, Satoshi Waguri3, Tatsuo Suganuma4, Yasuo Uchiyama3 and Kazuhisa Nakayama1,{ddagger}

1 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
2 Laboratory of Molecular Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan
3 Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
4 Department of Anatomy, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan



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  		Fig. 1. Localization of endogenous ARFRP1. (A) HeLa cells were fixed and double-stained for ARFRP1 and either the AP-1 {gamma}-adaptin subunit, GGA3, golgin-245 or GM130 followed by Cy3-conjugated anti-mouse IgG and Alexa Fluor 488-conjugated anti-rabbit IgG. Bar, 20 µm. (B) Immunoblot analysis for ARFRP1 in mock transfected HEK293 and HeLa cells, and HeLa cells transfected with HA-tagged ARFRP1. An asterisk indicates the positions of endogenous ARFRP1 and a double asterisk indicates exogenously expressed ARFRP1-HA. (C) Cryo-thin sections of HeLa cells expressing GFP-CI-MPR were double-labeled with mouse anti-GFP and rabbit anti-ARFRP1 (arrows) antibodies, which were then detected by two secondary antibodies conjugated with 15 nm- and 10 nm-colloidal gold particles, respectively. Arrowheads indicate two clathrin-coated vesicles, one of which contains a GFP-CI-MPR signal. Go, Golgi stack. Bar, 0.1 µm.

 


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  		Fig. 2. Effects of BFA on the localization of ARFRP1. HeLa cells were treated with 5 µg/ml BFA for 0, 2, 5, or 10 minutes and processed for indirect immunofluorescence analysis as described under Materials and Methods. Cells were double-stained with affinity-purified anti-ARFRP1 antibodies and monoclonal antibody to either the {gamma}-adaptin subunit of the AP-1 complex or golgin-245, followed by Cy3-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG. Bar, 20 µm.

 


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  		Fig. 3. Localization of Golgi proteins in cells overexpressing ARFRP1(T31N). HeLa cells expressing HA-tagged ARFRP1(T31N) (A-D; left column) were fixed and double-stained (right column) for HA and either golgin-97 (A), TGN46 (B), galactosyl transferase (GalT; C) or GGA3 (D) followed by Cy3-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-rabbit IgG (A,B) or anti-mouse IgG (C,D). Bar, 20 µm.

 


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  		Fig. 4. Arl1(Q71L) suppresses redistribution of golgin-97 induced by ARFRP1(T31N) expression. HeLa cells co-expressing a combination of either HA-tagged Arl1(WT) (A,B) or Arl1(Q79L) (C) and either FLAG-tagged ARFRP1(Q79L) (A) or ARFRP1(T31N) (B,C) were triple-stained for HA (middle column), FLAG (left column) and golgin-97 (right column) followed by Alexa Fluor 488-conjugated anti-rat IgG, Cy3-conjugated anti-mouse IgG and Cy5-conjugated anti-rabbit IgG. Bar, 20 µm.

 


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  		Fig. 5. Retrograde transport of TGN38 in cells expressing ARFRP1 mutants. (A) HeLa cells co-expressing FLAG-TGN38 and either HA-ARFRP1(WT), HA-ARFRP1(Q79L) or HA-ARFRP1(T31N) were incubated with mouse monoclonal anti-FLAG M2 antibody on ice for 50 minutes, washed, and further incubated at 37°C for 60 minutes. The cells were then fixed and stained with rat anti-HA and rabbit anti-ß-COP antibodies followed by Alexa Fluor 488-conjugated anti-rat, Cy3-conjugated anti-mouse and Cy5-conjugated anti-rabbit secondary antibodies. Bar, 20 µm. (B) The efficiency of the retrograde transport of TGN38 was estimated by calculating the ratio of the perinuclear overlapped area of the TGN38- and ß-COP-positive structures versus the total area of the perinuclear ß-COP-positive structures. In each case, the areas were estimated for over 10 cells.

 


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  		Fig. 6. Stx1 transport in cells expressing ARFRP1 mutants. HeLa cells expressing HA-tagged ARFRP1(T31N) (A,B) or ARFRP1(Q79L) (C,D) were incubated with Cy3-conjugated Stx1 at 19.5°C for 50 minutes, washed, and directly fixed (A,C) or shifted to 37°C for 60 minutes (B,D) then fixed, and stained with rat anti-HA and mouse anti-GalT antibodies followed by Alexa Fluor 488-conjugated anti-rat and Cy5-conjugated anti-mouse IgGs. Bar, 20 µm.

 


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  		Fig. 7. VSVG-GFP transport in cells expressing ARFRP1 mutants. (A) HeLa cells coexpressing GFP-tagged VSVG tsO45 and either HA-tagged ARFRP1(WT), ARFRP1(Q79L) or ARFRP1(T31N) were incubated at 40°C overnight and then shifted to 32°C for 30 minutes or 1 hour, and stained with rat anti-HA antibody followed by Cy3-conjugated anti-rat IgG. Asterisks indicate cells expressing ARFRP1-HA. (B) HeLa cells were transfected as described in A and incubated at 40°C overnight and then shifted to 32°C for 1 hour. The cell surface proteins were then biotinylated as described in Materials and Methods. Biotinylated surface proteins recovered with streptavidin-agarose beads (top panel) and total cell lysates (one twentieth amount of input; middle and bottom panels) were subjected to immunoblotting with anti-GFP (top and middle panels) or anti-HA antibody (bottom panel). (C) The band intensity in the top panel in B was estimated by Image Gauge software. This is representative of two independent experiments.

 

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© The Company of Biologists Ltd 2005