A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane Francesca De Marchis • Andrea Pompa • Roberta Mannucci • Tomas Morosinotto • Michele Bellucci Received: 29 May 2010 / Accepted: 21 July 2010 / Published online: 18 August 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowl- edge 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 recom- binant protein to the thylakoid membrane. The fusion pro- tein 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, Dzeolin, 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 Dzeolin, 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. Keywords Chloroplast transformation Á Disulfide bonds Á Protein folding Á Signal peptide Á Thylakoid Introduction Plastome (i.e. plastid genome) genetic engineering is a research area of plant biotechnology where significant advances have been accomplished (for review see Grevich and Daniell 2005; Bock 2007). Despite of this progress, the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in plastids is still quite limited. When a heterologous gene is inserted into the plastome and the corresponding protein is synthesized in the stroma, the correct folding of this protein is very uncertain in the new environment. Several post-transla- tional modifications take place in the stroma, such as multimerization (Daniell 2006), lipidation (Glenz et al. 2005), N-terminal methionine excision (Meinnel et al. 2006), but still they do not assure the correct folding of a specific recombinant protein. For example, a number of proteins require the formation of intramolecular or inter-chain disulfide bonds for their stability and/or their catalytic activity as enzymes. Disulfide linkage is also important for the immunogenicity of recombinant proteins to be used as vaccine antigens. Even though recombinant disulfide-bond containing proteins can be successfully expressed in transformed plastids, both in the stroma (Staub et al. 2000; Francesca De Marchis and Andrea Pompa contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9676-6) contains supplementary material, which is available to authorized users. F. De Marchis Á A. Pompa Á M. Bellucci (&) Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche (CNR), via della Madonna Alta 130, 06128 Perugia, Italy e-mail: michele.bellucci@igv.cnr.it R. Mannucci Istituto di Medicina Interna, Universita ` di Perugia, Perugia, Italy T. Morosinotto Dipartimento di Biologia, Universita ` degli Studi di Padova, 35121 Padova, Italy 123 Plant Mol Biol (2011) 76:427–441 DOI 10.1007/s11103-010-9676-6