Signal Peptide-Dependent Targeting of a Riceα-Amylase and Cargo Proteins to Plastids and Extracellular Compartments of Plant Cells

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Abstract
α-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether α-amylases are localized in plastids where starch is stored. To study the subcellular localization of α-amylases in plant cells, a rice (Oryza sativa) α-amylase, αAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of αAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated αAmy3 to be cotranslationally imported and processed in canine microsomes. αAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to αAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized αAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the αAmy3 SP was further studied by gain-of-function analyses. β-Glucuronidase (GUS) and green fluorescence protein fused with or without the αAmy3 SP was expressed in transgenic tobacco or rice. The αAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice α-amylase, αAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.