Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Abstract

The N-terminal proline-rich domain of ␥-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.The mechanisms by which prolamins, which lack the ER 3 retention (H/K)DEL motif, are retained and assembled in the ER-derived PBs are only partially understood. Different factors act as determinants of PB biogenesis, some of them derived from cis-cargo properties and others with a cellular trans-origin (1). It has been proposed that the physico-chemical properties of prolamins, such as hydrophobicity and disulfide bond formation, promote specific interactions resulting in the formation of large self-assemblies that are responsible for their retention in the ER and PB formation (2-4). These polymers could be excluded because of their size from being carried by the COP II vesicles that transport cargo proteins to the Golgi complex (5). However, the generic COPII vesicles are flexible enough for large cargo loading, including that of procollagen (more than 300 nm in size) (6) and the collagen VII trimer (900 kDa) (7).In maize, four distinct types of prolamins (␣-, ␤-, ␥-, and ␦-zein) coexist in the PBs (8) and play distinct roles in PB formation. Recently, maize storage protein mutants created through RNAi showed that ␥-RNAi maize mutant lines exhibited slightly altered protein body formation and that a more drastic effect was observed in the ␤-␥ combined mutant, where protein bodies showed an irregular shape, particularly in their periphery (9).Various studies on zein accumulation in heterologous expression systems suggest that ␥-zein and ␤-zein mediate t...

Protein body formation in the endoplasmic reticulum as an evolution of storage protein sorting to vacuoles: insights from maize γ-zein

“…However, the high importance of the first two N-terminal Cys in Zera-ECFP was not confirmed in full-length γ-zein: mutagenesis of these residues in Zera-ECFP resulted in full secretion (Llop-Tous et al, 2010), whereas 5C γ-zein is largely insoluble and ER-located, and it is almost unavailable for secretion. This indicates that the contribution of individual Cys residues of the N-terminal region to PB formation are less strict in full length γ-zein, as also suggested by domain deletion experiments (Geli et al, 1994).…”

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system

“…In maize, γ-zein contains six cystein residues, and N-terminal Cys 7 and Cys 9 are critical for oligomerization and PB formation. 10) Kawagoe reported that the formation of intermolecular disulfide bonds between the CCxQL motif and the PxxC motif of prolamin play an important role in polymerization in rice PB-Is. 18) On the other hand, 13b prolamin has no cystein residue, and 13b prolamin-GFP fusion proteins formed PB-Ilike structures in rice calli in our previous study.…”

Protein bodies: how the ER deals with high accumulation of recombinant proteins

“…Once PBs arise, they grow in size over time (Figure 1B), reaching their maximum size at 5 days post infiltration (dpi) for GFP‐ELP and GFP‐HFBI (Saberianfar et al ., 2015), and at 10 dpi for Zera®‐ECFP (Llop‐Tous et al ., 2010). In all cases, PB growth is accompanied by an increase in accumulation levels of the respective recombinant protein.…”

“…This strategy, however, does not work as well with Zera®, because the strong affinity of Zera® molecules to one another results in a condensed and sticklike alignment of Zera® molecules which excludes other molecules from the core of PBs (Llop‐Tous et al ., 2010). This was confirmed with co‐expression of secretory GFP or ER‐targeted GFP with Zera®‐DsRed in which the GFP signal localized to the periphery of Zera® PBs and not in their core, and explains why Zera®‐DsRed has no significant effect on EPO accumulation (Saberianfar et al ., 2016).…”

“…PBs induced by these processes are numerous, round and clustered, they remain in the ER, and they are not destined to the central vacuole (Llop‐Tous et al ., 2010; Saberianfar et al ., 2015, 2016). …”