title: The rice alpha-amylase glycoprotein is targeted from the Golgi apparatus through the secretory pathway to the plastids.
abstract: The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.
authors.affiliation: Graduate School of Science and Technology, Niigata University, Niigata, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kitajima
authors.forename: Satoru
authors.initials: S
authors.lastname: Asatsuma
authors.forename: Hisao
authors.initials: H
authors.lastname: Okada
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hamada
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kaneko
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nanjo
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawagoe
authors.forename: Kiminori
authors.initials: K
authors.lastname: Toyooka
authors.forename: Ken
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Masaki
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mitsui
mesh_terms: D000595:Amino Acid Sequence; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019697:Onions / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D013213:Starch; D000516:alpha-Amylases