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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

Hypothetical model for the import of the fluorescent fusion proteins and for PB formation in the plastid. (A) TP-DsZein likely enters the plastid via the TOC/TIC pathway (orange). After passing the TOC complex, it is probably recognized by the IMS translocation complex (blue) and further transported via the TIC channel into the stroma where the transit peptide (black) is removed and a PB (red) is formed. (B) ΔSP-DsZein and ΔSP-DsCRS may enter the plastid via the TOC complex or a non-canonical route. After crossing the outer plastid membrane the fusion protein assembles into insoluble polymers that are no longer competent for further transport. PBs are formed in the IMS and bud off from the plastid. The DsRed sequence is shown in red, γ-zein sequences are shown in purple, ribosomes are marked in brown.
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Figure 7: Hypothetical model for the import of the fluorescent fusion proteins and for PB formation in the plastid. (A) TP-DsZein likely enters the plastid via the TOC/TIC pathway (orange). After passing the TOC complex, it is probably recognized by the IMS translocation complex (blue) and further transported via the TIC channel into the stroma where the transit peptide (black) is removed and a PB (red) is formed. (B) ΔSP-DsZein and ΔSP-DsCRS may enter the plastid via the TOC complex or a non-canonical route. After crossing the outer plastid membrane the fusion protein assembles into insoluble polymers that are no longer competent for further transport. PBs are formed in the IMS and bud off from the plastid. The DsRed sequence is shown in red, γ-zein sequences are shown in purple, ribosomes are marked in brown.

Mentions: It is unclear whether the ΔSP-DsZein protein enters the plastid via the TOC/TIC complexes (Li and Chiu, 2010) or a non-canonical route (Armbruster et al., 2009). The TOC/TIC pathway involves hetero-oligomeric complexes in the inner and outer plastid membranes (Jarvis and Soll, 2002; Kessler and Schnell, 2009). Import via the TOC channel requires at least four different TOC proteins, all sharing conserved cysteine residues (Stengel et al., 2009). The formation and reduction of disulfide bonds between TOC proteins was shown to regulate this trafficking step (Kessler and Schnell, 2009), but there is no evidence for the formation of disulfide bonds between TOC components and incoming pre-proteins (Stengel et al., 2010). However, the cysteine residues in the γ-zein CRS are probably exposed and in a reduced form in the cytosol, so the formation of disulfide bridges with TOC components might also occur and prevent further transport through the TIC receptor. Instead, the protein would remain in the IMS, where apparently the conditions are appropriate to support the formation of PBs. In contrast, TP-DsZein is probably recognized by the IMS translocation complex and therefore further transported via the TIC channel (Figure 7). Interaction studies will be necessary to characterize the import pathways in more detail. It was not possible to regenerate stable transgenic plants transformed with the ΔSP-DsZein and TP-DsZein constructs (data not shown) suggesting that the formation of PBs in the plastids was not compatible with the regeneration of viable plants, perhaps because the PBs disturbed the sensitive redox equilibrium in the plastids (Wittenberg and Danon, 2008).


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)

Hypothetical model for the import of the fluorescent fusion proteins and for PB formation in the plastid. (A) TP-DsZein likely enters the plastid via the TOC/TIC pathway (orange). After passing the TOC complex, it is probably recognized by the IMS translocation complex (blue) and further transported via the TIC channel into the stroma where the transit peptide (black) is removed and a PB (red) is formed. (B) ΔSP-DsZein and ΔSP-DsCRS may enter the plastid via the TOC complex or a non-canonical route. After crossing the outer plastid membrane the fusion protein assembles into insoluble polymers that are no longer competent for further transport. PBs are formed in the IMS and bud off from the plastid. The DsRed sequence is shown in red, γ-zein sequences are shown in purple, ribosomes are marked in brown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Hypothetical model for the import of the fluorescent fusion proteins and for PB formation in the plastid. (A) TP-DsZein likely enters the plastid via the TOC/TIC pathway (orange). After passing the TOC complex, it is probably recognized by the IMS translocation complex (blue) and further transported via the TIC channel into the stroma where the transit peptide (black) is removed and a PB (red) is formed. (B) ΔSP-DsZein and ΔSP-DsCRS may enter the plastid via the TOC complex or a non-canonical route. After crossing the outer plastid membrane the fusion protein assembles into insoluble polymers that are no longer competent for further transport. PBs are formed in the IMS and bud off from the plastid. The DsRed sequence is shown in red, γ-zein sequences are shown in purple, ribosomes are marked in brown.
Mentions: It is unclear whether the ΔSP-DsZein protein enters the plastid via the TOC/TIC complexes (Li and Chiu, 2010) or a non-canonical route (Armbruster et al., 2009). The TOC/TIC pathway involves hetero-oligomeric complexes in the inner and outer plastid membranes (Jarvis and Soll, 2002; Kessler and Schnell, 2009). Import via the TOC channel requires at least four different TOC proteins, all sharing conserved cysteine residues (Stengel et al., 2009). The formation and reduction of disulfide bonds between TOC proteins was shown to regulate this trafficking step (Kessler and Schnell, 2009), but there is no evidence for the formation of disulfide bonds between TOC components and incoming pre-proteins (Stengel et al., 2010). However, the cysteine residues in the γ-zein CRS are probably exposed and in a reduced form in the cytosol, so the formation of disulfide bridges with TOC components might also occur and prevent further transport through the TIC receptor. Instead, the protein would remain in the IMS, where apparently the conditions are appropriate to support the formation of PBs. In contrast, TP-DsZein is probably recognized by the IMS translocation complex and therefore further transported via the TIC channel (Figure 7). Interaction studies will be necessary to characterize the import pathways in more detail. It was not possible to regenerate stable transgenic plants transformed with the ΔSP-DsZein and TP-DsZein constructs (data not shown) suggesting that the formation of PBs in the plastids was not compatible with the regeneration of viable plants, perhaps because the PBs disturbed the sensitive redox equilibrium in the plastids (Wittenberg and Danon, 2008).

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