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The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence.

Hofbauer A, Peters J, Arcalis E, Rademacher T, Lampel J, Eudes F, Vitale A, Stoger E - Front Bioeng Biotechnol (2014)

Bottom Line: Endogenous PBs are derived from the endoplasmic reticulum (ER).The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain.Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences , Vienna , Austria.

ABSTRACT
Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

No MeSH data available.


Related in: MedlinePlus

Protein body formation in the plastids (TP). (A) Confocal laser scanning microscopy. Protein bodies (arrowheads) within the plastids. Left: DsRed fluorescence, middle: plastid autofluorescence, right: merged channels. Note that in most cases one protein body can be observed per plastid. (B) Immunoelectron microscopy. Protein body (pb, arrowhead) within the stroma of the plastid (chl). Mitochondria (m). Bars = 5 μm (A) or 0.5 μm (B).
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Figure 4: Protein body formation in the plastids (TP). (A) Confocal laser scanning microscopy. Protein bodies (arrowheads) within the plastids. Left: DsRed fluorescence, middle: plastid autofluorescence, right: merged channels. Note that in most cases one protein body can be observed per plastid. (B) Immunoelectron microscopy. Protein body (pb, arrowhead) within the stroma of the plastid (chl). Mitochondria (m). Bars = 5 μm (A) or 0.5 μm (B).

Mentions: The formation of PBs was investigated by confocal microscopy 4–15 DPI. Construct SP-DsZein induced the formation of spherical bodies with a tendency to form clusters (Figure 3A) and electron microscopy confirmed the presence of PBs in the cytoplasm (Figure 3B), surrounded by a ribosome-studded membrane, indicating they had originated from the ER (Figure 3C). Construct TP-DsZein also induced the formation of spherical PBs but their distribution differed significantly from those induced by SP-DsZein. Confocal microscopy clearly revealed the formation of fluorescent PBs in the stroma (Figure 4). Most plastids contained a single PB in the stroma (Figure 4A). PBs formed by SP-DsZein are, like natural PBs of maize endosperm, in close contact to the inner face of the ER membrane (Figure 3B). Those formed by TP-DsZein are only in part, but not completely, in contact with thylakoids.


The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence.

Hofbauer A, Peters J, Arcalis E, Rademacher T, Lampel J, Eudes F, Vitale A, Stoger E - Front Bioeng Biotechnol (2014)

Protein body formation in the plastids (TP). (A) Confocal laser scanning microscopy. Protein bodies (arrowheads) within the plastids. Left: DsRed fluorescence, middle: plastid autofluorescence, right: merged channels. Note that in most cases one protein body can be observed per plastid. (B) Immunoelectron microscopy. Protein body (pb, arrowhead) within the stroma of the plastid (chl). Mitochondria (m). Bars = 5 μm (A) or 0.5 μm (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263181&req=5

Figure 4: Protein body formation in the plastids (TP). (A) Confocal laser scanning microscopy. Protein bodies (arrowheads) within the plastids. Left: DsRed fluorescence, middle: plastid autofluorescence, right: merged channels. Note that in most cases one protein body can be observed per plastid. (B) Immunoelectron microscopy. Protein body (pb, arrowhead) within the stroma of the plastid (chl). Mitochondria (m). Bars = 5 μm (A) or 0.5 μm (B).
Mentions: The formation of PBs was investigated by confocal microscopy 4–15 DPI. Construct SP-DsZein induced the formation of spherical bodies with a tendency to form clusters (Figure 3A) and electron microscopy confirmed the presence of PBs in the cytoplasm (Figure 3B), surrounded by a ribosome-studded membrane, indicating they had originated from the ER (Figure 3C). Construct TP-DsZein also induced the formation of spherical PBs but their distribution differed significantly from those induced by SP-DsZein. Confocal microscopy clearly revealed the formation of fluorescent PBs in the stroma (Figure 4). Most plastids contained a single PB in the stroma (Figure 4A). PBs formed by SP-DsZein are, like natural PBs of maize endosperm, in close contact to the inner face of the ER membrane (Figure 3B). Those formed by TP-DsZein are only in part, but not completely, in contact with thylakoids.

Bottom Line: Endogenous PBs are derived from the endoplasmic reticulum (ER).The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain.Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences , Vienna , Austria.

ABSTRACT
Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

No MeSH data available.


Related in: MedlinePlus