Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

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 (₄₆₈RVAAA₄₇₂) of cruciferin 1, an isoform of 12S globulins. The ₄₆₈RVA₄₇₀ motif forms a parallel β-sheet with the switch III residues (₁₂₇TMD₁₂₉) of VSR1-PA, and the ₄₇₁AA₄₇₂ motif docks to a cradle formed by the cargo-binding loop (₉₅RGDCYF₁₀₀), 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.

During seed development, storage proteins are deposited in a specialized organelle called the protein storage vacuole (PSV) and are mobilized to provide sources of carbon, nitrogen, and sulfur during germination (1). Seed storage proteins are synthesized as secretory proteins that are translocated into the endoplasmic reticulum (ER). How these proteins are transported to the PSV is not fully understood. In the receptor-mediated sorting pathway, storage proteins are sorted to the vacuoles via sequence-specific interactions with transmembrane sorting receptors (2). According to the latest model, sorting receptors could pick up the cargo proteins as early as in the ER and transport them through the Golgi apparatus to the trans-Golgi network (TGN), which then matures into the prevacuolar compartment (PVC) and PSV (2). Alternatively, storage proteins are concentrated and aggregated at the periphery of the cis-Golgi, where the dense vesicles (DVs) are formed (3, 4). DVs later bud off and fuse with the PVC, which matures into the PSV. Receptor–cargo interaction could play a role in the aggregation of storage proteins. For example, removal of the C-terminal hydrophobic (AFVY) residues of phaseolin, a storage protein of French bean (Phaseolus vulgaris), abolished aggregation of phaseolin and missorted it to the extracellular space (5).There are two families of sorting receptors, namely vacuolar sorting receptors (VSRs) and receptor-homology-transmembrane-RING-H2 (RMR) proteins (6–8). There are seven homologs of VSR and six homologs of RMR in the Arabidopsis thaliana genome. Unlike lysosomal sorting in animal cells that recognizes the posttranslational modification of mannose-6-phosphate (9), vacuolar sorting in yeast and plant cells is mediated by specific protein–protein interactions between the sorting receptors and the cargo proteins (6, 10–19). These sorting receptors recognize sequence-specific information, or vacuolar sorting determinants (VSDs), on the cargo proteins (20, 21). There are two types of VSD, the sequence-specific VSD (ssVSD) and the C-terminal VSD (ctVSD) (22–24). VSRs can recognize both ssVSDs and ctVSDs (6, 15, 25), while RMRs can only recognize ctVSDs (26, 27). ssVSD, often found in acidic hydrolases targeting lytic vacuoles, contains an NPIR motif with the consensus sequence of (N/L)-(P/I/L)-(I/P)-(R/N/S) (28). Mutations in the NPIR motif disrupt receptor–cargo interactions and lead to missorting of cargo proteins (18, 21, 29, 30). Unlike ssVSD that is located at internal sequence positions, ctVSD is only found at the C terminus of cargo proteins. No consensus sequence has been identified for ctVSD, but it is usually rich in hydrophobic residues (20). For example, the AFVY motif at the C terminus of phaseolin was found to be essential for targeting seed proteins to the PSV (31).VSRs are type I transmembrane proteins that contain a protease-associated (PA) domain, a central domain, and three epidermal growth factor (EGF) repeats in the luminal N-terminal region, followed by a single transmembrane domain (TMD) and a C-terminal cytoplasmic tail (Fig. 1A) (6, 12, 32). The PA domain and central domain are involved in sequence-specific interactions with ssVSDs (21, 33). We have previously determined the crystal structure of the PA domain of vacuolar sorting receptor isoform 1 (VSR1-PA) in complex with the ssVSD of barley aleurain (21) and showed that the PA domain is responsible for recognizing the sequences preceding the NPIR motif. Cargo binding induces the C-terminal tail to undergo a swivel motion that could relocate the central domain to cooperate with the PA domain for ssVSD recognition (21). The EGF repeats have unclear functions, but they might regulate the cargo binding by calcium-dependent conformational change of the PA and central domains (33, 34).Open in a separate windowFig. 1.Crystal structure of VSR1-PA in complex with the C-terminal pentapeptide (₄₆₈RVAAA₄₇₂) of CRU1. (A) Domain organization of VSRs. VSR1-NT consists of a protease-associated domain, a central domain, and three EGF repeats. sp, signal peptide. (B) Pull-down assay. E. coli–expressed VSR1-PA was incubated with NHS-resins coupled with the C-terminal peptide sequence of CRU1 (YRVAAA) or with glycine. A tyrosine residue was added to the N terminus of the peptide to facilitate the quantification of peptide concentration using A₂₈₀. After extensive washing to ensure VSR1-PA was not present in the last wash fractions (W), VSR1-PA bound (B) to the resins was analyzed by immunoblot with a VSR1-PA antibody. (C) Cartoon representation of the crystal structure of VSR1-PA in complex with the CRU1 C-terminal sequence, ₄₆₈RVAAA₄₇₂ (yellow). Switch I, II, and III regions and the cargo-binding loop are color-coded green, magenta, salmon, and cyan, respectively. The complex structure determined at pH 6.5 is shown. (D) A close-up view of the detailed receptor–cargo interactions. ₄₆₈RVA₄₇₀ of CRU1 forms a parallel β-sheet with ₁₂₈TMD₁₂₉ of switch III. The last two residues of CRU1, ₄₇₁AA₄₇₂, dock into a cradle formed by the conserved residues in the cargo-binding loop, ₉₅RGDCYF₁₀₀. The backbone conformation of the bound cargo is maintained by a number of backbone–backbone hydrogen bonds (dotted lines). The C-terminal carboxyl group of CRU1 forms salt bridges with Arg95 in the cargo-binding loop. Intermolecular hydrogen bonds and salt bridges are summarized (Right). (E) VSR1-PA undergoes conformational changes upon binding of ₄₆₈RVAAA₄₇₂. In the apo form of VSR1-PA (light blue), the cargo-binding site is occupied by switch III residues, where Glu133 forms salt bridges with Arg95. Cargo binding displaces the switch III residues away. N-terminal residues 20 to 24 that form strand β-1N in the apo form became disordered, making room for Asn46 in the switch II region to move toward and make a hydrogen bond with Met128 of switch III. Switch I residues (25 to 27) straighten up to form an antiparallel β-sheet with β-2. (F) Sequence of VSR1-PA, pumpkin PV72, pea BP-80, and soybean and French bean VSRs were aligned using the program MUSCLE (57). Secondary structure elements of the bound and apo forms of VSR1-PA are indicated above and below the alignment, respectively. Dotted lines indicate residues that are disordered in the crystal structures. Residues are numbered according to the VSR1-PA sequence.The role of VSRs in sorting seed storage proteins has been supported by genetic studies in Arabidopsis. In a pioneer study, Shimada and coworkers showed that the vsr1 knockout mutant missorted the seed storage proteins 12S globulin and 2S albumin to the extracellular space in Arabidopsis seeds (15). Zouhar and coworkers further showed that vsr1vsr3 and vsr1vsr4 double mutants reduced the amount of the mature form of 12S globulin in the PSV, suggesting that VSR1, VSR3, and VSR4 are the sorting receptors for 12S globulin (35). Moreover, tagging the C-terminal 24 residues of β-conglycinin to the C terminus of a secretory green fluorescent protein (GFP) was sufficient to target the fluorescent protein to the PSV in Arabidopsis seeds, while the fluorescent protein was missorted to the extracellular space in the vsr1 mutant (19). Since VSR1 can bind to the C-terminal sequence of both cruciferin 1 (CRU1), an isoform of 12S globulin, and β-conglycinin (15, 19), it is likely that VSR1 recognizes the sorting determinants in these sequences and sorts them to the PSV in seeds.The molecular mechanism of how VSRs recognize the sorting determinants of cargo proteins remains elusive. In this study, we report the crystal structure of the PA domain of VSR1 in complex with the C-terminal pentapeptide (₄₆₈RVAAA₄₇₂) of CRU1. Structural insights into receptor–cargo interaction were supported by mutagenesis and functional studies, which showed that a specific recognition between the VSR and ctVSD is essential for vacuolar sorting.