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Regulation of Golgi structure and function by ARF-like protein 1 (Arl1)

Lei Lu, Heinz Horstmann, Cheepeng Ng and Wanjin Hong

Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore



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  		Fig. 1. Enrichment of Arl1 on vesicular-tubular structures on the trans-side of the Golgi. Cryosections derived from CHO cells were labeled with Arl1 antibodies (large arrowheads) followed by 9 nm gold-particle-conjugated protein A (upper). Cryosections were also double-labeled with Arl1 antibodies followed by 14 nm gold particle-conjugated protein A (large arrowheads) as well as a monoclonal antibody against {gamma}-adaptin followed by 9 nm gold particle-conjugated protein A (small arrowheads) (lower). Bars: 100 nm. G, Golgi.

 


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  		Fig. 2. Saturable association of Arl1 with the Golgi. Control CHO cells (A-C) were processed for double labeling to reveal endogenous Arl1 and GS28. Pooled Arl1-transfected (D-F) or Arl1-EGFP transiently transfected CHO cells (G-J) were fixed and double labeled with a monoclonal antibody against GS28 (E,H) and a limiting amount of Arl1 antibodies to reveal the exogenous expressed Arl1 (D,G). Arrows indicate the Golgi marked by GS28 (D,E). Bars: 10 µm.

 


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  		Fig. 3. Golgi association of Arl1 depends on its N-terminal myristoylation. CHO cells were transiently transfected with constructs expressing Arl1(G2A)-EGFP in which the N-terminal myristoylation site Gly at position 2 was mutated into Ala, (A-C) or EGFP-Arl1 in which the myristoylation site was blocked by fusing Arl1 to the C-terminus of EGFP, (D-F). Cells were then processed to reveal the EGFP fusion protein as well as endogenous GS28. In contrast to Arl1-EGFP, these myristoylation-defective Arl1 mutants were not associated with the Golgi. Bars: 10 µm.

 


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  		Fig. 4. The GDP-bound form of Arl1 disassembles the Golgi apparatus. CHO (A-C) or A431 cells (D-L) were transiently transfected with a construct expressing Arl1(T31N) (A-C and G-L) or Arl1(T31N)-EGFP (D-F), in which Thr at position 31 was mutated to Asn. Cells were then processed for double labeling using a limiting amount of Arl1 antibody (A, G and J) and antibodies against GS28 (B), GT(ß-1,4-galactosyltransferase) (E), {gamma}-adaptin (H) or p115 (K). The Golgi labeling of GS28 and GT were disrupted in cells overexpressing Arl1(T31N) (A-F). {gamma}-adaptin (AP-1) was dissociated from perinuclear Golgi structure in Arl1(T31N)-expressing cells (G-I). Overexpression of Arl1(T31N) did not affect the Golgi-like distribution of p115, a Golgi-matrix protein (J-L). Bars: 10 µm.

 


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  		Fig. 5. Expression of the GTP-bound form of Arl1 caused an expansion of the Golgi. Pooled Arl1(Q71L) transfected CHO cells were double labeled with a limiting amount of Arl1 antibody to label exogenous Arl1(Q71L) and antibody against GS28 (A-C). Arl1(Q71L) was associated with Golgi (A) and caused an expansion of Golgi apparatus marked by GS28 (B). CHO cells were transiently transfected with constructs expressing Arl1(Q71L)-EGFP (D-F) or Arl1(G2A, Q71L)-EGFP (G-I). Cells were then processed to reveal expressed protein as well as endogenous GS28. In contrast to Arl1(Q71L)-EGFP, Arl1(G2A, Q71L)-EGFP was not detected in the Golgi apparatus and had no effect on the Golgi marked by GS28 (H). Bars: 10 µm.

 


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  		Fig. 6. Arl1(Q71L) causes massive Brefeldin A (BFA)-resistant Golgi recruitment of COPI coat (ß-COP). Arl1(Q71L) stably transfected CHO cells were treated with 10 µg/ml Brefeldin A for 0 minutes (A-C), 5 minutes (D-F) and 30 minutes (G-I) and then double labeled with a limiting amount of Arl1 antibody to reveal expressed Arl1(Q71L) and with monoclonal antibody against ß-COP. Bars: 10 µm.

 


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  		Fig. 7. Arl1(Q71L) causes massive Brefeldin A (BFA)-resistant Golgi recruitment of AP-1 coat ({gamma}-adaptin). Pooled Arl1(Q71L) transfected CHO cells were treated with 10 µg/ml Brefeldin A for 0 minutes (A-C), 10 minutes (D-F) and 30 minutes (G-I) and then double labeled with a limiting amount of Arl1 antibody to reveal expressed Arl1(Q71L) and a monoclonal antibody against {gamma}-adaptin. Bars: 10 µm.

 


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  		Fig. 8. Arl1(Q71L) causes massive Brefeldin A (BFA)-resistant Golgi recruitment of Golgi ARFs. Pooled Arl1(Q71L)-transfected CHO cells were treated with 10 µg/ml Brefeldin A for 0 minutes (A-C), 5 minutes (D-F) and 60 minutes (G-I) and then double labeled with a limiting amount of Arl1 antibody to reveal expressed Arl1(Q71L) and with a monoclonal antibody against Golgi ARFs. Bars: 10 µm

 


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  		Fig. 9. Transport of VSV-G to the cell surface is inhibited at the Golgi in Arl1(Q71L)-expressing cells. Pooled Arl1(Q71L)-transfected (A-C), Arl1(Q71L)-EGFP (D-F) or Arl1(G2A, Q71L)-EGFP (G-I) transiently transfected CHO cells were infected with VSVts045 at 32°C for one hour and followed by two hours in the absence and an hour in the presence of 10 µg/ml cycloheximide. Cells were fixed and processed to reveal overexpressed proteins (A, D and G) and VSV-G (B, E and H). Bars: 10 µm.

 


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  		Fig. 10. Overexpression of Arl1(Q71L) transforms the Golgi into an extensive vesicular-tubular network. Pooled Arl1(Q71L)-transfected CHO cells were infected with VSVts045 at 32°C for one hour and followed by two hours in the absence and one hour in the presence of 10 µg/ml cycloheximide. Cryosections derived from these cells were processed for immunogold EM to detect Arl1(Q71L) and VSV-G. In cells expressing moderate levels of Arl1(Q71L) (upper panel), Golgi cisternae became more dilated and expanded. 9 nm and 14 nm gold particles represent Arl1(Q71L) and VSV-G (arrowheads), respectively. In cells expressing high levels of Arl1(Q71L) (lower panel), the Golgi apparatus was transformed into an extensive tubular-vesicular network. Arl1(Q71L) (9 nm gold particles, small arrowheads) decorated the entire network, with VSV-G (14 nm gold particles, large arrowheads) being accumulated in the structure. Bars: 100 nm.

 


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  		Fig. 11. Ar11 and ARF1 have common and unique effectors. (A) Using Arl1(Q71L) as the bait in a yeast two-hybrid screening of human brain cDNA library, 18 interacting clones were identified and the results of DNA sequencing of these 18 clones are shown. (B) Interactions were assayed by mating of GAL4-BD/AH109 yeast cells with their respective GAL4-AD/Y187 yeast cells and by growing the resulting diploid yeast cells on the selective medium. The diploid yeast cells were assayed on a QDO plate containing X-{alpha}-gal. ‘-’ indicates no growth on the QDO plate after four days; ‘++’ indicates growth and blue coloration after four days; ‘+++’ indicates growth and strong blue coloration after four days. In yeast two- hybrid assays, POR1 interacted with the GTP form of both Arl1 and ARF1, whereas GGA1 interacted only with GTP form of ARF1. (C) An in vitro binding assay showing POR1 interaction with both Arl1 and ARF1 in guanine-nucleotide-dependent manner. 60 µg GDP- or GTP{gamma}S-exchanged GST-Arl1 or GST-ARF1 fusion proteins immobilized on glutathione sepharose beads were incubated with 100 µM of the appropriate guanine nucleotide and 10 µl of S35 Met labeled in vitro translated POR1 at 4°C over night. After washing, bound proteins were resolved by 12% SDS-PAGE and analyzed by the PhosphoImager. Upper panel: lane 1, 10% in vitro translated POR1; lane 2, GDP-exchanged GST-Arl1(T31N); lane 3, GTP{gamma}S-exchanged GST-Arl1(Q71L); lane 4, GDP-exchanged GST-ARF1; lane 5, GTP{gamma}S-exchanged GST-ARF1. Lower panel: the loading of each GST fusion protein.

 

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© The Company of Biologists Ltd 2001