title: The N-terminal cleavable pre-sequence encoded in the first exon of cystathionine γ-synthase contains two different functional domains for chloroplast targeting and regulation of gene expression.
abstract: Chloroplast transit peptide sequences (cTPs) located in the N-terminal region of nuclear-encoded chloroplast proteins are essential for their sorting, and are generally cleaved from the proteins after their import into the chloroplasts. The Arabidopsis thaliana cystathionine γ-synthase (CGS), the first committed enzyme of methionine biosynthesis, is a nuclear-encoded chloroplast protein. Arabidopsis CGS possesses an N-terminal extension region that is dispensable for enzymatic activity. This N-terminal extension contains the cTP and several functional domains including an MTO1 region, the cis-element for post-transcriptional feedback regulation of CGS1 that codes for CGS. A previous report suggested that the cTP cleavage site of CGS is located upstream of the MTO1 region. However, the region required for protein sorting has not been analyzed. In this study, we carried out functional analyses to elucidate the region required for chloroplast targeting by using a chimeric protein, Ex1:GFP, in which the CGS1 exon 1 coding region containing the N-terminal extension was tagged with green fluorescent protein. The sequence upstream of the MTO1 region was responsible for efficient chloroplast targeting and for avoidance of missorting to the mitochondria. Our data also showed that the major N-terminus of Ex1:GFP is Ala91, which is located immediately downstream of the MTO1 region, and the MTO1 region is not retained in the mature Ex1:GFP accumulated in the chloroplast. These findings suggest that the N-terminal cleavable pre-sequence harbors dual functions in protein sorting and in regulating gene expression. Our study highlights the unique properties of Arabidopsis CGS cTP among chloroplast-targeted proteins.
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan These authors contributed equally to this work. Present address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Hagiwara-Komoda
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan These authors contributed equally to this work. Present address: Chugai Pharmaceutical Co., Ltd., API Process Development Department, Tokyo, 115-8543 Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan Graduate School of Life Science, Hokkaido University, Sapporo, 060-0810 Japan.
authors.forename: Yui
authors.initials: Y
authors.lastname: Yamashita
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Onouchi
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan Graduate School of Life Science, Hokkaido University, Sapporo, 060-0810 Japan naito@abs.agr.hokudai.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Naito
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D008969:Molecular Sequence Data
chemical_list: C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases
keywords: Arabidopsis thaliana; Bifunctional sorting signal; Chloroplast transit peptide; Cystathionine γ-synthase; MTO1 region; Protein missorting