Open Access Research article

Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

Erin A Anthonio1, Chantal Brees1, Eveline Baumgart-Vogt2, Tsunaki Hongu3, Sofie J Huybrechts1, Patrick Van Dijck45, Guy P Mannaerts1, Yasunori Kanaho3, Paul P Van Veldhoven1 and Marc Fransen1*

Author Affiliations

1 Department of Molecular Cell Biology, Catholic University of Leuven, Herestraat 49, Leuven, Belgium

2 Institute for Anatomy and Cell Biology II, Justus-Liebig University, Aulweg 123, Giessen, Germany

3 Department of Physiological Chemistry, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba, Japan

4 Department of Molecular Microbiology, VIB, Leuven, Belgium

5 Department of Biology, Catholic University of Leuven, Kasteelpark Arenberg 31, Leuven, Belgium

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BMC Cell Biology 2009, 10:58  doi:10.1186/1471-2121-10-58

Published: 17 August 2009

Additional files

Additional file 1:

Phenotypic analysis of the oleate-grown Δarf1Δarf3 S. cerevisiae strain. Serial dilutions of wild-type (WT) yeast cells (strain BY4741) and yeast cells deficient in Arf1 (Δarf1), Arf3 (Δarf3), or Arf1 and Arf3 (Δarf1Δarf3) expressing EGFP-PTS1 were spotted onto plates with oleate as a sole carbon source. The plates were subsequently incubated at 30°C for five days. (A) Oleate consumption was scored by halo formation. (B) The subcellular distribution pattern of EGFP-PTS1 was visualized by fluorescence microscopy. The scale bar represents 5 μm. (C) The number of peroxisomes per cell was counted in randomly selected cells. The mean number of peroxisomes per cell is indicated by an asterisk. At least 150 oleate-grown cells were scored.

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Additional file 2:

Specificity of the monoclonal anti-Arf6 antibody. (A) Multiple sequence alignment of the rat Arf protein sequences. Amino acids identical in all aligned sequences are shown in red. Amino acid residues identical in five out of six sequences are shown in blue. Note that (i) Arf1 and Arf2 share the highest sequence identity (96% identical at the amino acid level; different amino acids are shaded in green), (ii) the latter protein is absent in humans, (iii) the amino acid sequences of Arf1, Arf3, Arf5 and Arf6 are completely identical in rat and human, and (iv) the amino acids not identical in rat and human Arf4 are highlighted in yellow. (B) Immunoblot analysis of equal amounts of extracts from CHO cells transfected with a monocistronic plasmid coding for Arf4-EGFP or a bicistronic plasmid encoding EGFP-PTS1 and no protein (-) or non-tagged human Arf1, Arf3, Arf5, or Arf6 proteins. The blots were probed with antibodies against EGFP (α-EGFP) or Arf6 (α-Arf6). Note that the expression levels of EGFP-PTS1 allow the indirect quantification of the Arf expression levels. The arrows indicate the migration of the full-length proteins. The arrowheads mark the Arf4-EGFP degradation products. The migration of relevant molecular mass markers (expressed in kDa) is shown at the left. (C) Immunoblot analysis of equal amounts of extracts from bacteria expressing (His)6-GST (H6-GST)-tagged human Arf proteins or a negative control protein (H6-GST-DCOH). The blots were probed with antibodies against (His)6 (α-H6) or Arf6 (α-Arf6). Note that, as – based on a Ponceau S staining – the expression levels of the H6-GST-tagged proteins varied greatly, the blots were cut into three pieces (each containing two conditions yielding similar amounts of recombinant protein) and incubated for different times in alkaline phosphatase-NBT/BCIP staining solution in order to normalize the signal intensities for equal amounts of recombinant protein. The arrows mark full-length proteins, the arrowheads point to degradation products.

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Additional file 3:

Arf6 ablation does not alter the localization of catalase in fetal mouse hepatocytes. Primary hepatocytes from mouse embryos (13.5 days) of Arf6+/+ and Arf6-/- littermates from control (-CF) and clofibrate-treated (+CF) pregnant Arf6+/- mice were isolated, seeded on collagen-coated cover glasses, cultured for 12 hours, and processed for indirect immunofluorescence microscopy with antibodies specific for catalase, a peroxisomal matrix protein. Scale bar: 20 μm.

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Additional file 4:

Effect of co-overexpression of Arf1T31N and Arf6T27N on peroxisomal protein import in Ptk2 cells. Ptk2 cells were transiently transfected with a plasmid coding for Arf1T31N-HA and a bicistronic plasmid encoding EGFP-PTS1 together with Arf6T27N. After 36 hours, the cells were fixed and processed for fluorescence analysis. The top row shows three merged images of the signals observed for Arf1T31N-HA (blue), EGFP-PTS1 (green), and endogenous Pex14p (red). The other rows represent enlarged views of the individual colour components of the areas shown in the insets. Note that the simultaneous expression of Arf6T27N (encoded by the same plasmid as EGFP-PTS1) and Arf1T31N has a strong influence on the localization of newly-synthesized EGFP-PTS1, but only a minor effect on the localization of endogenous Pex14p. Possible explanations for this apparent discrepancy are reviewed in the Results section of the main manuscript. Scale bar: 20 μm.

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Additional file 5:

Effect of co-overexpression of Arf1T31N and Arf6T27N on the appearance of Pex14p-immunoreactive particles in Ptk2 cells. Ptk2 cells were transiently transfected with a plasmid coding for Arf1T31N-HA and a bicistronic plasmid encoding EGFP-PTS1 together with Arf6T27N. After 36 hours, the cells were fixed and processed for fluorescence analysis. The top row shows three images of the signals observed for endogenous Pex14p (see Additional file 4, lower panels). The insets show an enlargement of the outlined regions. +, cell co-overexpressing Arf1T31N and Arf6T27N; -, cell overexpressing only Arf6T27N; 0, non-transfected cell (for overview images, see Additional file 4). Scale bar: 20 μm.

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