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First published online 20 November 2002
doi: 10.1242/jcs.00228

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Journal of Cell Science 116, 285-291 (2003)
doi: 10.1242/jcs.00228

Research Article

Differential usage of two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1

Sabrina M. Chabregas1, Douglas D. Luche2, Marie-Anne Van Sluys3, Carlos F. M. Menck2 and Marcio C. Silva-Filho1,*

1 Departamento de Genética, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Caixa Postal 83, 13400-970, Piracicaba, SP, Brazil
2 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
3 Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970, São Paulo, SP, Brazil

* Author for correspondence (e-mail: mdcsilva{at}esalq.usp.br)

Accepted 15 October 2002

Arabidopsis thaliana THI1 is encoded by a single nuclear gene and directed simultaneously to mitochondria and chloroplasts from a single major transcript. In vitro transcription/translation experiments revealed the presence of two translational products by the differential usage of two in-frame translational start codons. The coupling site-specific mutations on the THI1 encoding sequence with green fluorescent protein (GFP) gene fusions showed that translation initiation at the first AUG directs translocation of THI1 to chloroplasts. However, when translation starts from the second AUG, THI1 is addressed to mitochondria. Analysis of the translation efficiency of thi1 mRNA revealed that the best context for translation initiation is to use the first AUG. In addition, a suboptimal context in the vicinity of the second AUG initiation codon, next to a stable stem-and-loop structure that is likely to slow translation, has been noted. The fact that translation preferentially occurs in the first AUG of this protein suggests a high requirement for TH1 in chloroplasts. Although the frequency of upstream AUG translation is higher, according to the first AUG rule, initiation at the second AUG deviates significantly from Kozak's consensus. It suggests leaky ribosomal scanning, reinitiation or the internal entry of ribosomes to assure mitochondrial protein import.

Key words: Dual targeting, THI1, Mitochondria, Chloroplasts, Import, Translational regulation




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