The rice α-amylase glycoprotein is targeted from the golgi apparatus through the secretory pathway to the plastids

The well-characterized secretory glycoprotein, rice [Oryza sativa) α-amylase isoform I-1 (Amyl-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 Amyl-1 fused to green fluorescent protein (Amyl-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of Amyl-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 frans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with Amyl-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 Amyl-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 Amyl-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly354, located on the surface and on opposite sides of the Amyl-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.