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J. Biol. Chem., Vol. 279, Issue 41, 42445-42452, October 8, 2004

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Identification of the tRNA-binding Protein Arc1p as a Novel Target of in Vivo Biotinylation in Saccharomyces cerevisiae^*

Hyun Soo Kim, Ursula Hoja, Juergen Stolz, Guido Sauer¶, and Eckhart Schweizer||

From the Lehrstuhl für Biochemie der Universität Erlangen-Nürnberg, Erlangen D-91058, Lehrstuhl für Zellbiologie und Pflanzenphysiologie der Universität Regensburg, Regensburg D-93040, Germany, and the ^¶Institut für Klinische Molekularbiologie und Tumorgenetik der GSF, München D-81377, Germany

Biotin is an essential cofactor of cell metabolism serving as a protein-bound coenzyme in ATP-dependent carboxylation, in transcarboxylation, and certain decarboxylation reactions. The involvement of biotinylated proteins in other cellular functions has been suggested occasionally, but available data on this are limited. In the present study, a Saccharomyces cerevisiae protein was identified that reacts with streptavidin on Western blots and is not identical to one of the known biotinylated yeast proteins. After affinity purification on monomeric avidin, the biotinylated protein was identified as Arc1p. Using ¹⁴C-labeled biotin, the cofactor was shown to be incorporated into Arc1p by covalent and alkali-stable linkage. Similar to the known carboxylases, Arc1p biotinylation is mediated by the yeast biotin:protein ligase, Bpl1p. Mutational studies revealed that biotinylation occurs at lysine 86 within the N-terminal domain of Arc1p. In contrast to the known carboxylases, however, in vitro biotinylation of Arc1p is incomplete and increases with BPL1 overexpression. In accordance to this fact, Arc1p lacks the canonical consensus sequence of known biotin binding domains, and the bacterial biotin:protein ligase, BirA, is unable to use Arc1p as a substrate. Arc1p was shown previously to organize the association of MetRS and GluRS tRNA synthetases with their cognate tRNAs thereby increasing the substrate affinity and catalytic efficiency of these enzymes. Remarkably, not only biotinylated but also the biotin-free Arc1p obtained by replacement of lysine 86 with arginine were capable of restoring Arc1p function in both arc1 and arc1los1 mutants, indicating that biotinylation of Arc1p is not essential for activity.

Received for publication, June 25, 2004 , and in revised form, July 21, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 521/C7 (to J. S.) and Schw 19/31-2 (to E. S.). 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.

^|| To whom correspondence should be addressed: Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Staudtstrasse 5, Erlangen D-91058, Germany. Tel.: 49-9131-85-28-255; Fax: 49-9131-85-28-254; E-mail: eschweiz{at}biologie.uni-erlangen.de.

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