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

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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



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  		Fig. 1. Schematic representation of the chimeric gene constructs used for the in vitro transcription/translation experiments and functional in vitro analysis of the translation initiation context in the vicinity of the two potential in-frame AUG start codons in the thi1 mRNA. (A) The THI64-GUS construct retains both 5' thi1 in-frame ATGs intact codons. THI64{Delta}1-GUS contains a mutated first codon and an intact second codon. THI64{Delta}2-GUS presents an intact first ATG codon and a mutated second codon. THI64-1c-GUS contains a poor context introduced around the first ATG start codon. THI64-2c-GUS presents an optimum context inserted in the vicinity of the second in-frame ATG. In THI64-12c-GUS both initiation codons were altered (same modifications as described before). (B) Translational products from the first and second in-frame AUGs, 83 kDa and 75 kDa, respectively, of the chimeric gene constructions presented above (lanes 1 to 6) and SK+ GUS (67 kDa) used as a control. (C) Quantification of the translational products of the chimeric gene constructs. The GUS translation initiation efficiency was assumed to be similar in all situations. The intensity of the translational products is given as the ratio of the translation initiation in each of the two in-frame AUG start codons to that observed at the GUS initiation codon.

 


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  		Fig. 2. Schematic representation of the GFP fusions used in transient expression experiments. (a) The THI50-GFP construct corresponds to the N-terminus of THI1 (first 105 amino acids) containing the entire chloroplast transit peptide followed by 50 amino acids of the mature THI1 (which includes the putative mitochondrial presequence), with both 5' in-frame ATG codons. (b) THI50{Delta}1-GFP, the first ATG was mutated to ATC whereas the second in-frame start codon remained intact. (c) THI50{Delta}2-GFP, in which the second ATG was converted to ATC. (d) THImet2-GFP starts at the second ATG codon.

 


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  		Fig. 3. Transient expression of GFP fusions in wild-type tobacco leaf protoplasts. (A) Expression of the THI50-GFP construct. (B) Dual targeting to plastid and mitochondria by SYCO-GFP (Peeters et al., 2000Go). (C) Expression of the THI50{Delta}1-GFP construct. (D) Mitochondrial targeting of GFP mediated by ß-GFP construction (Duby et al., 2001Go). (E) Expression of the THImet2-GFP construct. (F) Expression of the THI50{Delta}2-GFP construct. (G) Plastid targeting of GFP by RecA-GFP (Köhler et al., 1997Go). The images were taken by epifluorescence microscope Axioplan 2. The fluorescence of GFP was observed at an excitation wavelength of 450-490 nm and emission wavelength of 520 nm (A-G, left panel). The autofluorescence of chloroplasts was observed at an excitation wavelength of 488 nm and emission wavelength of 664-696 nm (A-G, middle panel). The merged images of GFP and chlorophyll fluorescence are show in the right panel.

 


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  		Fig. 4. Sequence analysis of the 5'-end of thi1 mRNA. (A) Evidence for a stable stem-loop structure in a region between the two in-frame start codons (boxed). Predicted secondary structure of the 90 first codons of the wild-type thi1 mRNA. Structure was generated for 28°C by using the mfold version 2.3 program (http://mfold2.wustl.edu/~mfold/rna/for1-2.3.cgi). Black dots indicate G:C base pairs and grey dots indicate weaker A:U or G:U base pairing. (B) Complementary sequence between thi1 mRNA and 18S rRNA of A. thaliana. Vertical lines indicate base pairing and open circles represent G:U base pairing.

 




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