title: Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.
abstract: PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Endow
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Agostinho Gomes
authors.initials: AG
authors.lastname: Rocha
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Baldwin
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Roston
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Toshio
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan.
authors.forename: Hironari
authors.initials: H
authors.lastname: Kamikubo
authors.affiliation: Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, California, United States of America.
authors.forename: Kentaro
authors.identifier: http://orcid.org/0000-0001-5604-2486
authors.initials: K
authors.lastname: Inoue
mesh_terms: D002736:Chloroplasts / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D018532:Pisum sativum / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D010940:Plant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C011080:polyglycine; D012273:Ribulose-Bisphosphate Carboxylase