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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 bodies induced by ΔSP-DsCRS. (A) Bright field microscopy. Several large fluorescent protein bodies can be observed, some in close association with a plastid (arrowhead). (B) Immunoelectron microscopy, localization of DsRed. Large protein body in the vicinity of a plastid (chl). (C) CLSM image of protein bodies budding off a plastid (arrowheads). (D,E) Immunoelectron microscopy, localization of the fluorescent fusion protein in the IMS. Protein body budding off a plastid [(D), arrowhead, chl], showing tubular thylakoids close to the budding site [(D), arrow]. Abundant gold probes in the periphery of the plastid [(E), arrowhead]. (F) CLSM image of red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRed (far left), plastid autofluorescence (middle-left), GFP (middle-right), and merged channels (far right) are shown. Bars = 10 μm (A), 0.5 μm (B,D–F), or 5 μm (C).
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Figure 6: Protein bodies induced by ΔSP-DsCRS. (A) Bright field microscopy. Several large fluorescent protein bodies can be observed, some in close association with a plastid (arrowhead). (B) Immunoelectron microscopy, localization of DsRed. Large protein body in the vicinity of a plastid (chl). (C) CLSM image of protein bodies budding off a plastid (arrowheads). (D,E) Immunoelectron microscopy, localization of the fluorescent fusion protein in the IMS. Protein body budding off a plastid [(D), arrowhead, chl], showing tubular thylakoids close to the budding site [(D), arrow]. Abundant gold probes in the periphery of the plastid [(E), arrowhead]. (F) CLSM image of red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRed (far left), plastid autofluorescence (middle-left), GFP (middle-right), and merged channels (far right) are shown. Bars = 10 μm (A), 0.5 μm (B,D–F), or 5 μm (C).

Mentions: This construct behaved similarly to ΔSP-DsZein, forming large and heterogeneous PBs that grew at different rates and in some cases reached up to 5 μm in diameter (Figures 2A,B and 6A–C). As with ΔSP-DsZein, a relevant fraction of the DsRed-fusion protein was soluble in the absence of reducing agent (Figure 2C). By 15 DPI, large and irregular PBs were observed, which appeared to be budding from the plastid surface (Figures 6C,D). These large fluorescent PBs could even be observed by bright field microscopy (Figure 6A). Electron microscopy revealed frequent associations between these PBs and the plastids, as described for ΔSP-DsZein, and smaller PBs could again be observed budding from the plastid envelope (Figure 6D). Occasionally, the fusion protein is distributed as electron dense material within the intermembrane space without forming a spherical PB (Figure 6E). Transient expression in the leaves of TOC-GFP tobacco plants again confirmed that the PBs were covered by the outer plastid membrane (Figure 6F).


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 bodies induced by ΔSP-DsCRS. (A) Bright field microscopy. Several large fluorescent protein bodies can be observed, some in close association with a plastid (arrowhead). (B) Immunoelectron microscopy, localization of DsRed. Large protein body in the vicinity of a plastid (chl). (C) CLSM image of protein bodies budding off a plastid (arrowheads). (D,E) Immunoelectron microscopy, localization of the fluorescent fusion protein in the IMS. Protein body budding off a plastid [(D), arrowhead, chl], showing tubular thylakoids close to the budding site [(D), arrow]. Abundant gold probes in the periphery of the plastid [(E), arrowhead]. (F) CLSM image of red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRed (far left), plastid autofluorescence (middle-left), GFP (middle-right), and merged channels (far right) are shown. Bars = 10 μm (A), 0.5 μm (B,D–F), or 5 μm (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Protein bodies induced by ΔSP-DsCRS. (A) Bright field microscopy. Several large fluorescent protein bodies can be observed, some in close association with a plastid (arrowhead). (B) Immunoelectron microscopy, localization of DsRed. Large protein body in the vicinity of a plastid (chl). (C) CLSM image of protein bodies budding off a plastid (arrowheads). (D,E) Immunoelectron microscopy, localization of the fluorescent fusion protein in the IMS. Protein body budding off a plastid [(D), arrowhead, chl], showing tubular thylakoids close to the budding site [(D), arrow]. Abundant gold probes in the periphery of the plastid [(E), arrowhead]. (F) CLSM image of red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRed (far left), plastid autofluorescence (middle-left), GFP (middle-right), and merged channels (far right) are shown. Bars = 10 μm (A), 0.5 μm (B,D–F), or 5 μm (C).
Mentions: This construct behaved similarly to ΔSP-DsZein, forming large and heterogeneous PBs that grew at different rates and in some cases reached up to 5 μm in diameter (Figures 2A,B and 6A–C). As with ΔSP-DsZein, a relevant fraction of the DsRed-fusion protein was soluble in the absence of reducing agent (Figure 2C). By 15 DPI, large and irregular PBs were observed, which appeared to be budding from the plastid surface (Figures 6C,D). These large fluorescent PBs could even be observed by bright field microscopy (Figure 6A). Electron microscopy revealed frequent associations between these PBs and the plastids, as described for ΔSP-DsZein, and smaller PBs could again be observed budding from the plastid envelope (Figure 6D). Occasionally, the fusion protein is distributed as electron dense material within the intermembrane space without forming a spherical PB (Figure 6E). Transient expression in the leaves of TOC-GFP tobacco plants again confirmed that the PBs were covered by the outer plastid membrane (Figure 6F).

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