How cargo proteins are recognized and sorted to the vacuoles?

Abstract

In plant cells, cargo proteins destined to the vacuoles contain sequence specific information known as vacuolar sorting determinant (VSD) that is recognized and sorted by vacuolar sorting receptors (VSRs) to the vacuoles. To understand how VSR recognizes its cargos, our group have determined the crystal structures of the PA domain of Arabidopsis VSR1 in complex with the vacuolar sorting determinants (VSDs) of a protease aleurain [1] and a 12 S globulin cruciferin (CRU) 1 [2]. Structural comparison with the apo-form of VSR1-PA suggests that cargo-binding induces conformational changes in four regions of VSR1-PA. The VSDs bind to the cargo-binding loop consisting of a conserved motif of 95RGDCYF100. The invariant Arg-95 residue was found to be essential for receptor-cargo interactions and vacuolar sorting of cargo proteins. In vitro pull-down assay showed that VSR1 interacts with the C-terminal decapeptide sequences of CRU1, CRU4 and vicilin-like seed storage protein (VL) 22, but not with those of CRU2, CRU3, VL21 and VL43. Tagging the C-terminal sequences of CRU1 and VL22 to the secretary red fluorescent proteins are sufficient in redirecting the proteins to the vacuoles in Arabidopsis protoplasts. A P453A substitution converts the C-terminal sequence of CRU2 to a C-terminal VSD that is recognized by VSR1. Structural insights on receptor-cargo obtained could be useful in targeting recombinant proteins to protein storage vacuoles in seeds. This work was supported by grants from the Research Grants Council of Hong Kong Special Administrative Region, China (CUHK14151416, C4041-18EF, AoE/M-05/12, AoE/M403/16, and C4033-19E) and The National Natural Science Foundation of China (91854201 and31670179) and by direct grants from The Chinese University of Hong Kong. [1] Luo F. et al. (2014) “How vacuolar sorting receptor proteins interact with their cargo proteins: crystal structures of apo and cargo-bound forms of the protease-associated domain from an Arabidopsis vacuolar sorting receptor.” Plant Cell, 26, 3693-708 [2] Tsao H.E. et al. (2022) Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins. Proc Natl Acad Sci USA, 119, e2111281119.