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. 2009 Jun 15;185(6):1047–1063. doi: 10.1083/jcb.200811099

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© 2009 Rabl et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Fcj1 is required for normal mitochondrial morphology. (A) Wild-type (WT) and Δfcj1 cells expressing mitochondria-targeted GFP were grown on nonfermentable medium and visualized by fluorescence microscopy. (B) Domain structure of Fcj1 depicting the mitochondrial-targeting sequence (MTS), the transmembrane segment (TM), the coiled-coiled domain, and the conserved C-terminal domain (CTD) with corresponding positions of amino acid residues. (C) Subcellular fractionation of wild-type cells: mitochondria (Mito), microsomes (ER), and cytosol. Equal amounts of protein (50 µg) were analyzed by Western blotting with the indicated marker proteins: Tim44 (Mito), Erp1 (ER), and Hxk1 (Cytosol). (D) Submitochondrial localization of Fcj1. Wild-type mitochondria and mitoplasts generated by hypotonic swelling (SW) were treated with PK. f, specific proteolytic fragment of Oxa1. (E) Membrane association of Fcj1. Wild-type mitochondria were extracted with NaCl or Na carbonate. Membrane-bound (P) and soluble (S) fractions were loaded and analyzed by Western blotting using the indicated marker proteins. DLD, d-lactate dehydrogenase. (F) Homotypic interaction of Fcj1. Mitochondria from cells expressing a His-tagged (Fcj1-His₆) or a TAP-tagged variant of Fcj1 (Fcj1-TAP) or both were subjected to TAP affinity chromatography. Total (T; 10%), bound (B; 100%), and unbound (UB; 10%) material was analyzed by Western blotting with the indicated antibodies. Tom40 and TAP-tagged Fcj1 were detected simultaneously using rabbit antibodies against Tom40. Bars, 1 µm.