A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane

Abstract

Abstract The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD α--zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD α--zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this α--zein reveals a putative mutation alaine->valine (Ala->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 α--zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 α--zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 α--zein and the fl2 α--zein (Val->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 α--zein (Val->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 α--zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.