A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.
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 alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->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 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-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 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-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.
Gillikin
J W
JW
Department of Botany, North Carolina State University, Raleigh 27695, USA.
Zhang
F
F
Coleman
C E
CE
Bass
H W
HW
Larkins
B A
BA
Boston
R S
RS
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D003313:Zea mays / Q000235:genetics / Q000378:metabolism; D015028:Zein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D015028:Zein