Limits...
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-DsZein. (A) Immunoelectron microscopy. Gold particles decorating a protein body (pb) in close association with a plastid (chl). (B) CLSM image showing the accumulation of fluorescent fusion protein in the periphery of a plastid, as well as several budding sites (arrowhead). DsRed fluorescence (top), autofluorescence of plastids (middle), and merged channels (bottom) are shown. (C) Immunoelectron microscopy, localization of DsRed. Abundant gold probes are visible in the periphery of a plastid, showing a budding protein body (arrowhead). Note the tubular thylakoids in the vicinity of the budding site (arrows). (D) Detection of unsaturated lipids, by electron microscopy. Budding protein bodies are confined by the outer membrane (arrowheads) and the inner envelope membrane (double arrow) of the plastid. Tubular thylakoids can be observed at the budding site (arrows). (E) CLSM image showing red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRED (top), GFP (middle) and merged channels (bottom) are shown. Bars = 0.5 μm (A,D), 5 μm (B,E), or 0.25 μm (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263181&req=5

Figure 5: Protein bodies induced by ΔSP-DsZein. (A) Immunoelectron microscopy. Gold particles decorating a protein body (pb) in close association with a plastid (chl). (B) CLSM image showing the accumulation of fluorescent fusion protein in the periphery of a plastid, as well as several budding sites (arrowhead). DsRed fluorescence (top), autofluorescence of plastids (middle), and merged channels (bottom) are shown. (C) Immunoelectron microscopy, localization of DsRed. Abundant gold probes are visible in the periphery of a plastid, showing a budding protein body (arrowhead). Note the tubular thylakoids in the vicinity of the budding site (arrows). (D) Detection of unsaturated lipids, by electron microscopy. Budding protein bodies are confined by the outer membrane (arrowheads) and the inner envelope membrane (double arrow) of the plastid. Tubular thylakoids can be observed at the budding site (arrows). (E) CLSM image showing red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRED (top), GFP (middle) and merged channels (bottom) are shown. Bars = 0.5 μm (A,D), 5 μm (B,E), or 0.25 μm (C).

Mentions: The transient expression of a construct lacking an N-terminal signal sequence (ΔSP-DsZein) induced the formation of large PBs (~3 μm in diameter) that appeared to be localized mostly in close association with the plastids (Figure 5A). Some plastids were characterized by an irregular red fluorescent periphery, suggesting that smaller PBs were budding from the plastid membrane (Figure 5B). Indeed, electron microscopy revealed several PBs budding from the plastid surface (Figures 5C,D). Interestingly, tubular thylakoids [typically a sign of plastid stress; (Monselise et al., 1984)] were observed in the vicinity of these budding sites (Figures 5C,D). Detailed images revealed that recombinant protein accumulating at the plastid periphery was actually confined by two membranes, one enclosing the PB on the outside and the other defining the stromal border, indicating that the recombinant protein is localized in the intermembrane space (IMS), between the inner and outer membranes of the plastid envelope (Figure 5D). On several instances the PB protrudes from the chloroplast, suggesting a process of budding from the intermembrane space (Figures 5C,D). Lipid staining revealed that the budding PBs are enclosed in a lipid membrane derived from the outer membrane of the plastid envelope (Figure 5D).


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-DsZein. (A) Immunoelectron microscopy. Gold particles decorating a protein body (pb) in close association with a plastid (chl). (B) CLSM image showing the accumulation of fluorescent fusion protein in the periphery of a plastid, as well as several budding sites (arrowhead). DsRed fluorescence (top), autofluorescence of plastids (middle), and merged channels (bottom) are shown. (C) Immunoelectron microscopy, localization of DsRed. Abundant gold probes are visible in the periphery of a plastid, showing a budding protein body (arrowhead). Note the tubular thylakoids in the vicinity of the budding site (arrows). (D) Detection of unsaturated lipids, by electron microscopy. Budding protein bodies are confined by the outer membrane (arrowheads) and the inner envelope membrane (double arrow) of the plastid. Tubular thylakoids can be observed at the budding site (arrows). (E) CLSM image showing red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRED (top), GFP (middle) and merged channels (bottom) are shown. Bars = 0.5 μm (A,D), 5 μm (B,E), or 0.25 μm (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Protein bodies induced by ΔSP-DsZein. (A) Immunoelectron microscopy. Gold particles decorating a protein body (pb) in close association with a plastid (chl). (B) CLSM image showing the accumulation of fluorescent fusion protein in the periphery of a plastid, as well as several budding sites (arrowhead). DsRed fluorescence (top), autofluorescence of plastids (middle), and merged channels (bottom) are shown. (C) Immunoelectron microscopy, localization of DsRed. Abundant gold probes are visible in the periphery of a plastid, showing a budding protein body (arrowhead). Note the tubular thylakoids in the vicinity of the budding site (arrows). (D) Detection of unsaturated lipids, by electron microscopy. Budding protein bodies are confined by the outer membrane (arrowheads) and the inner envelope membrane (double arrow) of the plastid. Tubular thylakoids can be observed at the budding site (arrows). (E) CLSM image showing red fluorescent protein bodies enclosed by the outer plastid envelope membrane highlighted with a TOC-GFP membrane marker (arrowheads). DsRED (top), GFP (middle) and merged channels (bottom) are shown. Bars = 0.5 μm (A,D), 5 μm (B,E), or 0.25 μm (C).
Mentions: The transient expression of a construct lacking an N-terminal signal sequence (ΔSP-DsZein) induced the formation of large PBs (~3 μm in diameter) that appeared to be localized mostly in close association with the plastids (Figure 5A). Some plastids were characterized by an irregular red fluorescent periphery, suggesting that smaller PBs were budding from the plastid membrane (Figure 5B). Indeed, electron microscopy revealed several PBs budding from the plastid surface (Figures 5C,D). Interestingly, tubular thylakoids [typically a sign of plastid stress; (Monselise et al., 1984)] were observed in the vicinity of these budding sites (Figures 5C,D). Detailed images revealed that recombinant protein accumulating at the plastid periphery was actually confined by two membranes, one enclosing the PB on the outside and the other defining the stromal border, indicating that the recombinant protein is localized in the intermembrane space (IMS), between the inner and outer membranes of the plastid envelope (Figure 5D). On several instances the PB protrudes from the chloroplast, suggesting a process of budding from the intermembrane space (Figures 5C,D). Lipid staining revealed that the budding PBs are enclosed in a lipid membrane derived from the outer membrane of the plastid envelope (Figure 5D).

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