A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane

Abstract

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…Plastid-encoded pre-Tic40 was properly processed and targeted and assembled into a functional complex at the inner membrane of chloroplast (Singh et al 2008). Zeolin with a plant secretory signal peptide was targeted to the thylakoid membrane upon chloroplast transformation (De Marchis et al 2011). Recombinant disulfide-bond containing proteins can be expressed in transformed plastids when targeted to the lumen of thylakoids (Bally et al 2008).…”

Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

“…Recently, it has been shown that a ER plant signal peptide is capable of carrying a fusion protein called zeolin, whose gene is inserted into the plastoma, to the thylakoid membranes, where the recombinant protein accumulates as trimers. In the stroma, the folding of the same protein without the signal peptide is hampered as the protein accumulates at low amounts in a monomeric form (De Marchis et al, 2011). Thus, we can speculate that whatever is the sorting mechanism that directs zeolin to the thylakoids, it probably also has a stabilizing effect on the recombinant protein (Fig.…”

“…The accumulation of phaseolin in the ER was much higher than that in the vacuole, both as phaseolin-KDEL (data not shown) and as zeolin (arrowhead), which forms large aggregates (vertical bar) due to the formation of interchain disulfide bonds (Mainieri et al, 2004). In the chloroplast (CHL) of transplastomic plants expressing the phaseolin fusion protein zeolin, the recombinant protein devoid of its signal peptide (Dzeo) was barely detectable in the stroma (str; lanes 5 and 6); conversely, when targeted to the thylakoid membrane (thy; lanes 7 and 8), it is clearly detectable (De Marchis et al, 2011). Molecular mass in kD (numbers at right) is indicated.…”

“…When the enzyme alkaline phosphatase, a homodimer with two intramolecular disulfide bonds for each subunit, necessary for stability and catalytic activity of the molecule, was expressed both in the stroma and in the thylakoid lumen, the results indicated that sorting to the thylakoid lumen leads to larger amounts and more active enzyme (Bally et al, 2008). In addition, the Cys residues of another recombinant protein, capable of forming interchain disulfide bonds, can be oxidized if addressed to the thylakoid membranes (De Marchis et al, 2011). Conversely, the same proteins when localized into the stroma are unable to form this type of covalent linkage.…”

Unsolved problems in plastid transformation

“…23,28 Post-translational modifications, such as disulphide bond formations, can influence the stability of recombinant proteins produced in plastids contributing to protein accumulation. 28 Moreover, correct folding of the proteins by formation of disulphide bonds is often required for functional tertiary and quaternary structures and, therefore, for antigenicity of the recombinant proteins expressed in transplastomic plants. 3 On the other hand, glycosylation is absent in plastids, preventing the production of glycoproteins.…”