期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：1
DOI：10.1073/pnas.2111281119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Seeds such as rice and soybean are major food staples in the human diet. During seed development, storage proteins are deposited in a specialized organelle called the protein storage vacuole and are mobilized to provide nutrients during germination. Storage proteins are transported as cargoes via specific protein–protein interactions with the vacuolar sorting receptors. Supported by structural and mutagenesis studies, our work provides insights into how the sequence-specific information, or the vacuolar sorting determinant, on the storage proteins is recognized by the vacuolar sorting receptors for their targeting to the vacuoles. Insights gained into the rules of receptor–cargo recognition will be useful in engineering recombinant proteins for biotechnological applications of the protein storage vacuoles in seeds.
In
Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (
468RVAAA
472) of cruciferin 1, an isoform of 12S globulins. The
468RVA
470 motif forms a parallel β-sheet with the switch III residues (
127TMD
129) of VSR1-PA, and the
471AA
472 motif docks to a cradle formed by the cargo-binding loop (
95RGDCYF
100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in
Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in
Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.
