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A Short Peptide in Rice Glutelin Directs Trafficking of Protein into the Protein Storage Vacuoles of the Endosperm Cells

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Abstract

Several protein vacuolar sorting determinants (VSDs) have been identified in higher plants. Glutelin as a major storage protein in rice endosperm cells is transported to a protein storage vacuole (PSV). How glutelin sort to PSV and the mechanism of the intracellular trafficking has remained unknown. Here, a sequence-specific vacuolar sorting determinant (ssVSD) is identified by serial deletions of rice glutelin and its role in the protein-sorting process analyzed by transgenic approaches and transient assays. The ssVSD consists of six residues (QRLKHN) within the β-subunit of glutelin is sufficient to direct the glutelin to the protein body II in the rice endosperm cells. We found that protein-sorting via the ssVSD takes place by a ∼680-kDa sorting complex containing the receptor Oryza sativa receptor-like membrane Ring-H2 3 (OsRMR3). Further study indicated that OsRMR3 and the ssVSD are essential for glutelin trafficking. Furthermore, site-directed mutagenesis showed that the leucine residues in the ssVSD are critical for protein sorting.

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Acknowledgments

We would like to thank Dr. Liwen Jiang for kindly providing the OsRMR1 and AtVSR1 antibodies, MVB and TGN marker plasmids. This work was granted by the Projects of the National Natural Science Foundation of China (no. 30671286) and the National High-Tech R&D Program (863 Program) of China (no. 2011AA100604).

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  1. Key State Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences and Center of Engineering Research of Plant Biotechnology and Germplasm Utilization, Ministry of Education, Wuhan University, Wuhan, 430072, People’s Republic of China

    Wen Li, Tingting Xie, Qinchuan Qiu, Tingting Ning & Daichang Yang

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  1. Wen Li
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Correspondence to Daichang Yang.

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Supplemental Table 1

Vacuolar sorting determinants (VSD) found in higher plants (JPEG 23 kb)

High-resolution image (TIFF 2,069 kb)

Supplemental Figure 1

Supporting information for the different constructs. a Schematic diagram of the different truncated fragments from the glutelin β-subunit expressed in the different transgenic lines. b The hydrophobicity of the glutelin β-subunit as determined with MacVector software (version: 11.0.4). c The amino acid sequence of the glutelin β-subunit (JPEG 31 kb)

High-resolution image (TIFF 1,163 kb)

Supplemental Figure 2

Diagram of the protein sorting and trafficking routes in the rice endosperm cells. The red circles represent the proteins without VSD or with the ER retention signal (K/HDEL); the green circles represent the proteins containing VSD; the yellow circles represent the proteins containing secretion signals and orange unregulated circles indicate VSD-specific receptors, such as OsRMR3 (JPEG 17 kb)

High resolution image (TIFF 572 kb)

Supplemental Figure 3

Immuno-electron micrographs (IEM) of transgenic endosperm cells expressing OsrHSA. b and d Partial magnifications of the marked squares of the micrographs of a and c. The location of the gold particles indicates that OsrHSA is ultimately destined to the cytosolic space as shown in a and b. c and d The ribosome studded endoplasmic reticulum membrane (ERM), where OsrHSA is synthesized (JPEG 80 kb)

High-resolution image (TIFF 2,770 kb)

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Li, W., Xie, T., Qiu, Q. et al. A Short Peptide in Rice Glutelin Directs Trafficking of Protein into the Protein Storage Vacuoles of the Endosperm Cells. Plant Mol Biol Rep 31, 1492–1505 (2013). https://doi.org/10.1007/s11105-013-0624-x

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