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J. Biol. Chem., Vol. 283, Issue 13, 8564-8572, March 28, 2008

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Topology of Amphipathic Motifs Mediating Golgi Localization in ArfGAP1 and Its Splice Isoforms^*

From the Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

The interaction of the Arf1-directed GTPase-activating protein ArfGAP1 with the Golgi apparatus depends on motifs in its noncatalytic part that are unstructured in solution but are capable of folding into amphipathic helices in vitro upon interaction with poorly packed lipids. In previous studies a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and in vitro folding features of the region encompassing the amphipathic motifs (residues 199-294). Point mutation analysis revealed that most hydrophobic residues within this region contribute to Golgi localization, whereas analysis by proline replacements and alanine insertions revealed that Golgi interaction depends on folding into two amphipathic helices with a short interrupting sequence. Analysis of splice isoforms containing 10-residue in-frame insertions within their first amphipathic motifs revealed that the insertion causes a truncation of the amphipathic helix that does not extend beyond the insertion sequence. Lastly, a lysine replacement mutant recently reported to bind to negatively charged liposomes in a curvature-independent manner showed normal cellular distribution, suggesting that Golgi targeting of Arf-GAP1 may involve factors other than sensing lipid packing.

Received for publication, November 28, 2007 , and in revised form, January 10, 2008.

^* This work was supported by Israel Science Foundation Grant 448/04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Fax: 9724-8225153; E-mail: danc{at}tx.technion.ac.il.

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