GDP-restricted Arl1T31N abolishs Golgi association of Golgin-97 and Golgin-245 but not GM130 and p115. Arl1T31N was expressed transiently in A431 cells. Cells were then processed for the double-labeling with a limiting amount of Arl1 antibody (A, D, G, and J) and Mab against Golgin-97 (B), Golgin-245 (E), GM130 (H), or p115 (K). Bar, 10 μm.

Knockdown of Arl1 levels by siRNA dissociates Golgin-97 and Golgin-245 from the Golgi apparatus. (A) Hela cells were transfected with indicated siRNA. After 48 h, the total cell lysates were subjected to SDS-PAGE and immunoblotting analysis to detect Arl1 and β-tubulin. (B) Hela cells were transfected with Arl1 siRNA (D–F and J–O) or control siRNA (A–C and G–I). After 48 h, cells were double-labeled with Arl1 antibody (left panels) and antibodies against Golgin-97 (B and E), Golgin-245 (H and K), or GM130 (panel N). Bar, 10 μm.

Golgi dissociation of Golgin-97 and Arl1 are temporally coupled in response to brefeldin A (BFA) treatment. Hela cells were treated with 10 μg/ml brefeldin A for 0 min (A–C and M–O), 5 min (D–F and P–R), 10 min (G–I), and 30 min (J–L). Cells were then processed for double-labeling using Arl1 antibody (A, D, G, and J) and Golgin-97 Mab (B, E, H, and K), or double-labeling using Arl1 antibody (M and P) and Mab anti-ARFs (N and Q). Bar, 10 μm.

GTP-restricted Arl1 recruits Golgin-97 onto the Golgi apparatus in a switch II region-dependent manner. A431 cells were transiently transfected with expressing plasmid for Arl1Q71L (A–F) or Arl1SIIm (G–L). The resulting cells were either not treated (A–C and G–L) or treated with 10 μg/ml brefeldin A for 60 min (D–F) and then processed for double-labeling using a limiting amount of Arl1 antibody that selectively detect overexpressed protein (A, D, G, and J) and Mab against Golgin-97 (B, E, and H) or p115 (K). Bar, 10 μm.

The switch II region of Arl1 is necessary for interaction with GRIP domains. (A) Alignment of N-terminal 93 residues of rat Arl1 with the corresponding regions of other Arl1 as well as rat and yeast ARF1. Identical residues are colored red. Residues conserved in four of the six proteins were highlighted with a green background. The point mutation mutants of Arl1Q71L are indicated above. The switch II region of Arl1 is underlined and Arl1SIIm has its switch II region replaced by that of ARF1, whereas ARF1SIIm has its switch II region replaced by that of Arl1. (B) The various Arl1Q71L mutants were tested for interaction with the GRIP domain of Golgin-97, Golgin-245, or the full-length POR1 by the yeast two-hybrid assay.

Ectopic targeting of Arl1Q71L but not ARF1Q71L onto endosomes results in selective recruitment of Golgin-97 onto the endosomal membrane. A431 cells were transiently transfected with expressing construct for Myc-SNX3 (A–C), Myc-SNX3-Arl1Q71L (D–F′l and J–R), or Myc-SNX3-ARF1Q71L (G–I′). Cells were double-labeled with antibody against Myc to reveal the exogenously expressed SNX3 or its fusion proteins (left panels) and antibodies against Golgin-97 (B, E, E′, H, and H′), p115 (K), GT (N and N′), or β-COP (Q). Images D′–F′, G′–I′, and M′–O′ are the indicated x-z section of the x-y images of D–F, G–I, and M–O, respectively. Bar, 10 μm.

Conserved residues of the GRIP domain are important for interaction with Arl1 as well as for Golgi targeting. (A) Alignment of the amino acid sequences of four GRIP domains of human (prefix h) and Drosophila (prefix d) Golgins defines several conserved residues of the GRIP domains. The most conserved residues are indicated in red with a yellow background. The intermediate conserved residues are shown in blue under light blue background and the residues conserved in only some GRIP domains were highlighted with green background. The positions of point mutations analyzed in this study are indicated above the sequence of human Golgin-97. (B) Interaction of various forms of the Golgin-97 GRIP domain with Arl1Q71L as analyzed by ONPG β-galactosidase assay using the yeast two-hybrid system. (C) Interaction of various forms of Golgin-97 GRIP domain with Arl1Q71L as analyzed by cell growth in the yeast two-hybrid system. (D) The indicated forms of Golgin-97 GRIP domain mutants tagged with EGFP were expressed in transfected NRK cells (top row) and colabeled antibody against GM130 (Texas Red, middle row). There exists a strong correlation between interaction with Arl1Q71L and Golgi targeting of these mutant GRIP domains. Bar, 10 μm.

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.