Skip to main content
. 2003 Sep;14(9):3767–3781. doi: 10.1091/mbc.E03-01-0864

Figure 1.

Figure 1.

GTP-restricted Arl1 interacts with GRIP domains of Golgins. (A) Arl1Q71L, Arl1T31N, ARF1Q71L, and ARF1T31N were tested respectively in the yeast two-hybrid assay for interaction with the GRIP domain of Golgin-97, Golgin-245, GCC1p, KIAA0336, the full-length POR1, or GGA1. (B) ONPG β-galactosidase assay shows different interaction strengths of Arl1Q71L with various GRIP domains, POR1, or GGA. (C) Golgin-97 full-length but not Golgin-97Δ GRIP interacts with Arl1-Q71L in yeast two-hybrid assay, showing the interaction is only through the GRIP domain of Golgin-97. (D) Hela cell cytosol was incubated with 60 μg of various immobilized recombinant proteins as indicated. Proteins retained by the beads were eluted by SDS sample buffer and subjected to immunoblot analysis with the indicated antibodies. Significant amounts of Golgin-245 (first panel) and Golgin-97 (second panel) were retained on GTPγ S (lane 4 and 6) but not GDP (lane 3 and 5) charged forms of GST-Arl1. As negative controls, GST (lane 2), various form of GST-ARF1 (lane 7 and 8) or GST-Arl4 (lane 9 and 10) failed to retain Golgin-245 and Golgin-97. The endosomal EEA1 (third panel) was not retained by any of these beads. The bottom panel is the coomassie blue stained SDS-PAGE gel showing quantity of the respective GST fusion proteins. Lane 1 represents a 3% cytosol loading control for Golgin-245, Golgin-97 and EEA1. (E) Arl1 (top panel) but not ARFs (middle panel, blotted with Mab 1D9) in Hela cell cytosol was efficiently retained by immobilized GST-GRIP domain of Golgin-245 (lane 3) but not GST (lane 2). Lane 1 shows the 5% cytosol loading control. The bottom panel is the coomassie blue stained SDS-PAGE gel showing quantity of the recombinant GST or fusion protein. (F) Various forms of Arl1 tagged with EGFP, together with EGFP, were expressed in transfected 293T cells. The resulting cell lysates were immunoprecipitated with antibodies against EGFP, and the immunoprecipitates were analyzed by immunoblotting to detect Golgin-245. Golgin-245 was selectively coimmunoprecipited with GTP-restricted Arl1Q71L-EGFP (lane 5). Lane 1 represents 3% cytosol input used for the immunoprecipitation, and the bottom panel shows the 5% various EGFP fusion proteins recovered after immunoprecipitation.