Relevant elements of a Maize γ-zein domain involved in protein body biogenesis
Llop-Tous, Immaculada (Centre de Recerca en Agrigenòmica)
Madurga, Sergio (Universitat de Barcelona. Departament de Química Física)
Giralt, Ernest (Institut de Recerca Biomèdica de Lleida)
Marzabal, Pablo (ERA Biotech)
Torrent, Margarita (Centre de Recerca en Agrigenòmica)
Ludevid, M. Dolors (Centre de Recerca en Agrigenòmica)

Data:	2010
Resum:	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) 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 and Cys) 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.
Ajuts:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2005/SGR-00182 Ministerio de Ciencia e Innovación BIO2008-00799
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Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Amino acid sequence motifs ; Cysteine residues ; Fluorescent protein ; Immunoelectron microscopy ; Molecular dynamics simulations ; Nicotiana benthamiana ; Proline-rich domains ; Site directed mutagenesis
Publicat a:	Journal of biological chemistry, Vol. 285, Issue 46 (November 2010) , p. 35633-35644, ISSN 1083-351X

DOI: 10.1074/jbc.M110.116285
PMID: 20829359

13 p, 4.1 MB

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