Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE - PubMed (original) (raw)

Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE

Akira Honda et al. J Cell Biol. 2005.

Abstract

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16-amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.

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Figures

Figure 1.

Figure 1.

Subcellular localization of Arf chimeras. (A) Numbering and secondary structural elements (α helices and β sheets) of human Arf-1 and Arf-6 are shown. Identical residues between Arf-1 and Arf-6 are shaded in gray. Residues marked in red indicate amino acids 101–116 and 97–112 of Arf-1 and Arf-6, respectively. (B, C, and E) COS-7 cells overexpressing GalT-GFP and HA-tagged Arf-1 (B), untagged Arf-6 (C), or untagged Arf-6-1-6 (E) were untreated (Unt) or incubated in the presence of 20 μg/ml nocodazole for 2 h (+Nz). Cells were fixed and immunolabeled with antibodies against HA (B) or Arf-6 (C and E) followed by Alexa 594 anti–mouse and anti–rabbit antibodies, respectively. Bars, 10 μm. (D) Diagrams of chimeras of Arf-1 and Arf-6 are shown. Blue and yellow regions represent Arf-1 and Arf-6 sequences, respectively. + and − indicate ability to localize to the Golgi.

Figure 2.

Figure 2.

BFA-induced redistribution of Arf-6-1-6 and Arf-1-6-1. COS-7 cells overexpressing GFP-fused Arf-1, Arf-6-1-6, or Arf-1-6-1 were untreated (Unt) or incubated in the presence of 5 μg/ml BFA for 2 min and fixed. Bar, 10 μm.

Figure 3.

Figure 3.

Identification of critical amino acids in residues 101–116 of Arf-1 for Golgi targeting of the Arf-6-1-6 chimera. (A) Sequence comparison of residues 101–116 of Arf-1 and corresponding residues of Arf-6 and mutants of Arf-6-1-6 are shown. Bold characters indicate residues unique to Arf-6. (B) COS-7 cells overexpressing GalT-GFP and the Arf constructs indicated were fixed and immunolabeled with antibodies against Arf-6 followed by Alexa 594 anti–rabbit antibodies. 100 cells were counted and the fraction of transfected cells that show clear colocalization of overexpressed Arf proteins with GalT-GFP was noted. The result shown is the mean ± SD for three experiments. (C) Sequence comparison of residues 101–116 of Arf-1 and corresponding residues of related proteins are shown. Bold characters indicate residues identical to 110M and 113E of Arf-1.

Figure 4.

Figure 4.

Amino acid residues 101–116 of Arf-1 are targeted to the Golgi. (A) Schematic representation of GFP-Arf-1(101–116). (B–D) HeLa cells expressing GFP-Arf-1(101–116) were fixed and immunolabeled with antibodies against β-COP (B and D) or GM130 (C) followed by Alexa 594 anti–rabbit and anti–mouse antibodies, respectively. Cells expressed GFP-Arf-1(101–116) at low to moderate level (B) or high level (C and D). The transfected cells in C and D are outlined. Images I and II in C show magnified views of boxed areas. Bars, 10 μm.

Figure 5.

Figure 5.

Arf-1, but not Arf-1-6-1, interacts and colocalizes with membrin. (A–D) His6-tagged Arf-1 and Arf-1-6-1, and Arf-1M110I, E113D were incubated with lysate from HeLa cells expressing myc-membrin in the presence of GDP. A cross-linking reagent DSP was added where indicated. Arfs were recovered and proteins were analyzed by immunoblotting. Shown is one experiment representative of three (A) or two (B) performed with similar results. In C and D, relative amount of membrin bound to His6-Arf for replicate experiments was determined using a densitometer. (E and F) HeLa (E) or NRK (F) cells overexpressing myc-membrin and Arf-1-GFP or Arf-1-6-1-GFP were fixed and immunolabeled with antibodies against myc followed by Alexa 594 anti–mouse antibodies. Bar, 10 μm. (F) Quantitative analysis of area overlap in the Golgi region for NRK cells as described in Materials and methods. Error bars are the mean ± SD.

Figure 6.

Figure 6.

Membrin “protects” Arf-1 from BFA-induced redistribution. (A–C) COS-7 cells overexpressing myc-membrin and Arf-1-GFP or Arf-1-6-1-GFP (A), myc-membrin and Arf-1-HA (B), or Arf-1-GFP and myc-rbet1 or HA-syntaxin5 (C) were untreated (Unt) or incubated in the presence of 5 μg/ml BFA for indicated times and fixed. Cells were immunolabeled with antibodies against myc (A), HA and β-COP (B), or myc or HA (C) followed by Alexa 594 anti–mouse (A and C), or Alexa 488 anti–mouse and Alexa 594 anti–rabbit (B) antibodies. Bars, 10 μm. (D and E) HeLa cells overexpressing Arf-1-GFP alone (D, control) or Arf-1-6-1-GFP alone (E, control), or with myc-membrin or myc-rbet1, or HA-syntaxin5 were untreated or incubated in the presence of 5 μg/ml BFA for 2 min and fixed and processed for immunolabeling with antibodies against myc or HA followed by Alexa 594 anti–mouse and anti–rabbit antibodies, respectively. To quantify Golgi and non-Golgi pools of Arf-1-GFP, one region of interest was drawn around Golgi and the other region of interest was drawn around the rest of the cell (representing total cellular fluorescence). The mean fluorescence intensity associated with the Golgi and total cell was measured for Arf-1-GFP. The total Arf-1-GFP fluorescence associated with the Golgi was expressed as a fraction of the total cellular fluorescence, and this procedure was repeated for 10 cells. Error bars are the mean ± SD.

Figure 7.

Figure 7.

Anterograde movement of membrin and Arf-1 into the Golgi in BFA-recovering cells. (A) NRK cells overexpressing myc-membrin and Arf-1-GFP or Arf-1-6-1-GFP were incubated in the presence of 5 μg/ml BFA for 30 min, washed free of BFA, and incubated in the absence of BFA for the indicated times of wash out (WO) and fixed. Cells were immunolabeled with antibodies against myc followed by Alexa 594 anti–mouse antibodies. (B) NRK cells overexpressing myc-membrin were treated as in A and fixed. Cells were immunolabeled with antibodies against myc and β-COP followed by Alexa 488 anti–mouse and Alexa 594 anti–rabbit antibodies. Bars, 10 μm.

Figure 8.

Figure 8.

Comparison of the distribution of Arf-1 and Arf-1-6-1 to Arf-GEFs. NRK cells overexpressing GFP-fused Arf-1, Arf-1-6-1, and Arf-1-6-1MXXE were fixed and immunolabeled with antibodies against GBF1 (A) or BIG1 (C) followed by Alexa 594 anti–mouse and anti–rabbit antibodies, respectively. Bars, 10 μm. (B and D) Quantitative analysis of area overlap in the Golgi region as in A and C. Error bars are the mean ± SD.

Figure 9.

Figure 9.

Arf-1 and Arf-1-6-1 are regulated differently at Golgi membranes. (A) The kinetics of recovery of GFP-fused Arf-1, Arf-1-6-1, and Arf-1-6-1MXXE in the Golgi region after photobleaching are shown. The Golgi regions of cells expressing Arfs-GFP were photobleached at maximal laser intensity. Immediately after the bleach, cells were scanned every 5 s. The fluorescence intensity in the bleach area was measured. Shown is one experiment representative of three performed with similar results. (B) Models for membrane association/dissociation cycle of Arf-1 and Arf-1-6-1. See text for details.

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