Preserving the functional integrity of BiP1 is essential for rice storage protein export from the endoplasmic reticulum

Rice seed storage proteins (SSPs) undergo synthesis within the endoplasmic reticulum (ER). However, our understanding of the biochemical transformations that SSPs experience before leaving the ER and the corresponding quality acceptance criteria is still limited. In this study, we isolated a new glutelin precursor over-accumulation (57H) mutant, ke17 , which is defective in the ER export of SSPs. This defect leads to a reduction in the size of protein body I (PBI) and protein body II (PBII) in the endosperm, as well as the fragmentation of PBIs. Molecular cloning revealed that the KE17 encodes a novel dominant mutant allele of BiP1 ( mbip1 ), a member of the Hsp70 protein family, with a deletion of two conserved amino acids Ile58 and Ala59 in the nucleotide-binding domain. Subcellular localization experiments indicated that the mutation of BiP1 did not affect the localization of the ER and PBI. Nevertheless, yeast two-hybrid and luciferase complementation assays demonstrated that mbip1 weakened the interactions with nucleotide-exchange factors (Fes1A, Fes1C) and co-chaperones (HOP1, HOP2). We propose that the 58-59 deletion disrupts the ATP-dependent conformational cycle of BiP1 without interfering with its ER targeting, thereby impairing substrate folding and forward storage protein trafficking to PBIIs and accumulation in ER-derived PBIs. This work established BiP1 as a crucial hub in rice endosperm protein body biogenesis. • A new 57H mutant ke17 was identified, which is defective in SSPs’ ER export and causes reduced sizes of PBI and PBII in endosperm as well as PBI fragmentation. • Map-based cloning showed KE17 as a novel dominant mutant allele of BiP1 . • Deletion of Ile58 and Ala59 does not disrupt BiP1 localization but selectively weakens its interaction with NEFs (Fes1A, Fes1C) and co-chaperones (HOP1 and HOP2).