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. 2012 Feb 6;196(3):327-35.
doi: 10.1083/jcb.201107115. Epub 2012 Jan 30.

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

Chantal Christis  1 , Sean Munro
Affiliations

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

Chantal Christis et al. J Cell Biol. .

Abstract

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

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Figures

Figure 1.
Figure 1.
Liposome-based method for isolating Arf family effectors. (A) A schematic of the liposome-based purification method. His10-tagged Arf is bound to light liposomes (i), mixed with cell lysate and heavy liposomes (intended to reduce nonspecific binding to the light liposomes; ii), overlayed with less dense layers (iii), and, after centrifugation, liposomes and bound proteins are separated from the rest of the lysate (iv). Liposomes were then washed by diluting in buffer and repeating step iv. (B) Proteins from lysates of Drosophila S2 cells that bound to liposomes coated with nucleotide-locked forms of Drosophila Arl1, Arf1, and Arf4 prepared as in A. Lanes contain material from liposomes (Li) or 1/200 of subnatant (Su) or input lysate (Ly). Gels were either stained with Coomassie blue or immunoblotted for Drosophila GCC88 (dGCC88) as indicated. Molecular mass is indicated in kilodaltons. (C) Comparison of the peptide spectral counts for proteins on liposomes coated with GTP- or GDP-locked Arf1. Coloring indicates known Arf1 effectors (red) and the known Arf1 GEF Garz (green). COP, coatomer protein. (D) Comparison of the peptide spectral counts for known Arf effectors on liposomes coated with GTP-locked Arf1 or GTP-locked Arf4. Proteins of the same complex are shown in the same color (coat proteins COPI in red, AP-1 in purple, AP-3 in yellow, and the coiled-coil Golgi microtubule–associated protein [GMAP] in blue). (C and D) The graphs are representative of two independent experiments.
Figure 2.
Figure 2.
Sec71 binds to Arl1-coated liposomes in a GTP-dependent manner. (A) Comparison of the peptide spectral counts for proteins bound to liposomes coated with GTP- or GDP-locked forms of ARFRP1. (B) As in A, except for Arl1. Known effectors dGolgin-245 and dGCC88 are shown (closed red circles) along with Sec71 (open red circles), and proteins among the top 100 found on liposomes are indicated in purple. (A and B) The graphs are representative of two independent experiments. (C) Anti-GFP immunoblot of a small-scale liposome-binding assay using lysates (Ly) of S2 cells expressing Sec71-GFP as input and the indicated forms of Arl1 or Arf1. Li, liposomes; Su, subnatant. Molecular mass is indicated in kilodaltons.
Figure 3.
Figure 3.
Sec71 and Arl1 colocalize on the trans-Golgi. (A) Confocal micrographs of S2 cells expressing GFP-tagged Sec71 and stained for dGM130 (cis-Golgi) or dGolgin245 (trans-Golgi). (B) Confocal micrographs of S2 cells coexpressing Sec71-GFP and Arl1-RFP and stained as in A. (A and B) Sec71 is closer to the dGolgin245 than to dGM130 but does not colocalize with the former. However, the significance of this is uncertain, as the antibody to dGolgin245 was raised to the N-terminal 200 residues of this 1,489-residue coiled-coil protein (Sinka et al., 2008) and so may be binding up to 200 nm from the membrane to which dGolgin245 is anchored. Bars, 5 µm.
Figure 4.
Figure 4.
The N terminus of Sec71 binds to the Golgi and Arl1-GTP. (A) A schematic of Sec71 showing the catalytic Sec7 domain and other regions conserved in orthologs. The DCB (dimerization and Cyp5 binding), HUS (homology upstream of Sec7), and HDS (homology downstream of Sec7) domains are as previously described (Mouratou et al., 2005; Bui et al., 2009). (B) Confocal micrographs of S2 cells expressing GFP-tagged truncations of Sec71-GFP shown in A, in some cases along with Arl1-RFP. Cells were stained with an antibody against dGM130. Bars, 5 µm. (C) Silver-stained protein gel showing the binding of a purified recombinant form of the N terminus of Sec71 to Ni-NTA liposomes coated with His-tagged Arl1 or Arf1. The indicated forms of the G protein were bound to Ni-NTA liposomes, after which purified Strep-tagged Sec71(N-453) was added. After a 1-h incubation, liposomes were floated through an OptiPrep gradient, and protein samples were prepared from the liposome (Li) and subnatant (Su; 16% of input). Molecular mass is indicated in kilodaltons.
Figure 5.
Figure 5.
Arl1 is required for recruitment of BIG1 and BIG2 to the Golgi. (A) Confocal micrographs of HeLaM cells treated with siRNA against Arl1 and mixed 50:50 with untreated cells before plating on slides for staining with antibodies to the indicated endogenous proteins. Representative Golgi regions in the boxed areas are shown magnified in the insets. In cells lacking Arl1, the GRIP domain protein golgin-245 or GCC88 is displaced from the Golgi as expected. In such cells, BIG1 and BIG2 are also displaced from the Golgi, whereas other Golgi proteins are apparently unaffected. Bars, 15 µm. (B) Confocal micrographs of z stack projections of fields of HeLaM cells treated with a nontargeting siRNA (control) or an Arl1 siRNA, stained for the indicated proteins, and imaged with identical settings. The ratio of BIG1 staining to GM130 staining was quantified for all the cells in the field (n > 60), and the difference between the control and two different Arl1 siRNAs is highly statistically significant (two-tailed unpaired t test). Error bars show the SEM. Bars, 50 µm. (C) As in A, except that the cells were stained for the indicated endogenous proteins to show that the cis-Golgi Arf GEF, GBF1, retains its Golgi localization when Arl1 is knocked down. Bars, 15 µm.
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