title: Relevant elements of a maize gamma-zein domain involved in protein body biogenesis.
abstract: The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.
authors.affiliation: Centre de Recerca en Agrigenòmica, Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: Immaculada
authors.initials: I
authors.lastname: Llop-Tous
authors.forename: Sergio
authors.initials: S
authors.lastname: Madurga
authors.forename: Ernest
authors.initials: E
authors.lastname: Giralt
authors.forename: Pablo
authors.initials: P
authors.lastname: Marzabal
authors.forename: Margarita
authors.initials: M
authors.lastname: Torrent
authors.forename: M Dolors
authors.initials: MD
authors.lastname: Ludevid
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*; D015028:Zein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins; D015028:Zein