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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

(A) Overview of the fluorescent signal distribution following transient expression with the constructs shown in Figure 1. (B) Protein body growth and size distribution after infiltration. Bars represent the standard error; n = 25. (C) Solubility of the recombinant fusion proteins. Leaf samples were extracted in PBS (soluble fraction) and pellets were then re-extracted under strong reducing conditions. (D) Immunoblot analysis of total protein extracts from leaf samples. Antiserum against the HIS-tag was used to detect the recombinant protein.
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Figure 2: (A) Overview of the fluorescent signal distribution following transient expression with the constructs shown in Figure 1. (B) Protein body growth and size distribution after infiltration. Bars represent the standard error; n = 25. (C) Solubility of the recombinant fusion proteins. Leaf samples were extracted in PBS (soluble fraction) and pellets were then re-extracted under strong reducing conditions. (D) Immunoblot analysis of total protein extracts from leaf samples. Antiserum against the HIS-tag was used to detect the recombinant protein.

Mentions: The N-terminal portion of γ-zein is necessary for the ability of this protein to form PBs in the ER (Geli et al., 1994). To investigate whether PBs can also be created in other compartments, we designed a fusion protein comprising DsRed and 89 residues of γ-zein starting from the fourth amino acid after the N-terminal signal peptide cleavage site, hereafter γ-zein(4–93). This is the same zein fragment that promotes the formation of ER-located PBs when fused at the C-terminus of the vacuolar storage protein phaseolin, in the chimeric protein zeolin (Mainieri et al., 2004). The fragment was the basis for different constructs targeting the fusion protein to the cytosol (ΔSP-DsZein), ER (SP-DsZein), and plastids (TP-DsZein). As control, we used a sequence that is similar to the hydrophobic domain in γ−zein(4–93) but lacks any cysteine residues, and thus lacks the ability to form PBs (SP-DsHR). An overview of these constructs is provided in Figure 1. PB formation was induced with all constructs except the negative control (Figure 2A). A comparison of PB biogenesis and growth in the different compartments indicated that PBs induced by the SP-DsZein and TP-DsZein constructs were limited to a size of 1–1.5 μm, which was achieved ~4 DPI, and showed a homogeneous size distribution. In contrast, the PBs induced by ΔSP-DsZein had an average diameter of ~3 μm and were heterogeneous in terms of size and growth rate (Figure 2B).


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)

(A) Overview of the fluorescent signal distribution following transient expression with the constructs shown in Figure 1. (B) Protein body growth and size distribution after infiltration. Bars represent the standard error; n = 25. (C) Solubility of the recombinant fusion proteins. Leaf samples were extracted in PBS (soluble fraction) and pellets were then re-extracted under strong reducing conditions. (D) Immunoblot analysis of total protein extracts from leaf samples. Antiserum against the HIS-tag was used to detect the recombinant protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A) Overview of the fluorescent signal distribution following transient expression with the constructs shown in Figure 1. (B) Protein body growth and size distribution after infiltration. Bars represent the standard error; n = 25. (C) Solubility of the recombinant fusion proteins. Leaf samples were extracted in PBS (soluble fraction) and pellets were then re-extracted under strong reducing conditions. (D) Immunoblot analysis of total protein extracts from leaf samples. Antiserum against the HIS-tag was used to detect the recombinant protein.
Mentions: The N-terminal portion of γ-zein is necessary for the ability of this protein to form PBs in the ER (Geli et al., 1994). To investigate whether PBs can also be created in other compartments, we designed a fusion protein comprising DsRed and 89 residues of γ-zein starting from the fourth amino acid after the N-terminal signal peptide cleavage site, hereafter γ-zein(4–93). This is the same zein fragment that promotes the formation of ER-located PBs when fused at the C-terminus of the vacuolar storage protein phaseolin, in the chimeric protein zeolin (Mainieri et al., 2004). The fragment was the basis for different constructs targeting the fusion protein to the cytosol (ΔSP-DsZein), ER (SP-DsZein), and plastids (TP-DsZein). As control, we used a sequence that is similar to the hydrophobic domain in γ−zein(4–93) but lacks any cysteine residues, and thus lacks the ability to form PBs (SP-DsHR). An overview of these constructs is provided in Figure 1. PB formation was induced with all constructs except the negative control (Figure 2A). A comparison of PB biogenesis and growth in the different compartments indicated that PBs induced by the SP-DsZein and TP-DsZein constructs were limited to a size of 1–1.5 μm, which was achieved ~4 DPI, and showed a homogeneous size distribution. In contrast, the PBs induced by ΔSP-DsZein had an average diameter of ~3 μm and were heterogeneous in terms of size and growth rate (Figure 2B).

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