Tài liệu Báo cáo khoa học: The most C-terminal tri-glycine segment within the polyglycine stretch of

The most C-terminal tri-glycine segment within the

polyglycine stretch of the pea Toc75 transit peptide plays

a critical role for targeting the protein to the chloroplast

outer envelope membrane

Amy J. Baldwin and Kentaro Inoue

Department of Plant Sciences, College of Agricultural & Environmental Sciences, University of California, CA, USA

Most proteins found in plastids are encoded in the

nuclear genome, translated on cytosolic ribosomes with

cleavable N-terminal transit peptides, and imported

into the organelles post-translationally. The translocon

at the outer envelope membrane of chloroplasts 75

(Toc75) is postulated to function as a general protein

translocation channel [1–4], and was also shown to be

involved in targeting of a signal-anchored outer envel￾ope membrane protein [5]. Toc75 appears to be enco￾ded by a single functional gene in Arabidopsis thaliana

[6] and its disruption by a T-DNA insertion caused an

embryo-lethal phenotype [7], indicating the essential

role of Toc75 in the viability of plants.

Unlike other proteins destined for the outer mem￾branes of chloroplasts or mitochondria, which do not

require cleavable targeting sequences [8–10], Toc75 is

synthesized with an N-terminal transit peptide that

consists of two domains (Fig. 1) [11]. The first part

behaves as a typical stromal targeting sequence [12],

and is removed by a stromal processing peptidase

Keywords

chloroplast protein translocation channel;

polyglycine; protein targeting; transit

peptide; tripeptide segment

Correspondence

K. Inoue, Department of Plant Sciences,

College of Agricultural & Environmental

Sciences, University of California, One

Shields Avenue, Davis, CA 95616, USA

Fax: +1 530 752 9659

Tel: +1 530 752 7931

E-mail: [email protected]

(Received 18 January 2006, accepted

13 February 2006)

doi:10.1111/j.1742-4658.2006.05175.x

The protein translocation channel at the outer envelope membrane of

chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal

transit peptide. Within the transit peptide of the pea Toc75, a major por￾tion of the 10 amino acid long stretch that contains nine glycine residues

was shown to be necessary for directing the protein to the chloroplast outer

membrane in vitro [Inoue K & Keegstra K (2003) Plant J 34, 661–669]. In

order to get insights into the mechanism by which the polyglycine stretch

mediates correct targeting, we divided it into three tri-glycine segments and

examined the importance of each domain in targeting specificity in vitro.

Replacement of the most C-terminal segment with alanine residues resulted

in mistargeting the protein to the stroma, while exchange of either of the

other two tri-glycine regions had no effect on correct targeting. Further￾more, simultaneous replacement of the N-terminal and middle tri-glycine

segments with alanine repeats did not cause mistargeting of the protein as

much as those of the N- and C-terminal, or the middle and C-terminal seg￾ments. These results indicate that the most C-terminal tri-glycine segment

is important for correct targeting. Exchanging this portion with a repeat of

leucine or glutamic acid also caused missorting of Toc75 to the stroma. By

contrast, its replacement with repeats of asparagine, aspartic acid, serine,

and proline did not largely affect correct targeting. These data suggest that

relatively compact and nonhydrophobic side chains in this particular region

play a crucial role in correct sorting of Toc75.

Abbreviations

mtHsp70, mitochondrial heat shock protein 70; Plsp1, plastidic type I signal peptidase 1; psToc75, Toc75 from Pisum sativum; SPP, stromal

processing peptidase; Toc, translocon at the outer envelope membrane of chloroplasts.

FEBS Journal 273 (2006) 1547–1555 ª 2006 The Authors Journal compilation ª 2006 FEBS 1547