Protein Targeting into the Complex Plastid of Cryptophytes Sven B. Gould, 1 Maik S. Sommer, 1 Katalin Hadfi, 1 Stefan Zauner, 1 Peter G. Kroth, 2 Uwe-G. Maier 1 1 Cell Biology, Philipps-University Marburg, Karl-von-Frisch Straße 8, 35042 Marburg, Germany 2 Department of Biology, University of Konstanz, Universita¨tsstraße 10, 78457 Konstanz, Germany Received: 25 April 2005 / Accepted: 25 July 2005 [Reviewing Editor: Dr. Yves Van de Peer] Abstract. The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost mem- branes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different bio- chemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precur- sor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topo- genic signal of one protein localized in the periplas- tidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins. Key words: Secondary plastids — Periplastidal compartment — Nucleomorph — Protein transport — Cryptophytes Introduction Plastids of land plants and green and red algae as well as of glaucocystophytes are surrounded by a double membrane, the plastid envelope (Martin et al. 2002; Cavalier-Smith 2003; McFadden and van Dooren 2004). Import of proteins into these so-called primary plastids, is accomplished in land plants by two trans- locons termed Toc and Tic ( translocators of the outer and inner chloroplast membrane, respectively), with the specificity for unidirectional transport of nucleus- encoded plastid proteins (reviewed in Soll and Schleiff 2004). Secondary or complex plastids are surrounded by one or two additional membranes and have evolved by the engulfment and subsequent reduction of a photo- trophic alga containing primary plastids (secondary endosymbiosis [see Cavalier-Smith 2000; McFadden 2001; Prechtl and Maier 2002]). Some important algal groups harbor complex organelles, such as the peridi- nin-containing dinoflagellates and the phototrophic euglenoids with plastids surrounded by three mem- branes or heterokont algae, haptophytes, and the apicomplexa with four-membrane bounded plastids (Stoebe and Maier 2002, Cavalier-Smith 2003). Moreover, cryptophytes and chlorarachniophytes, both also possessing four-membrane bounded plast- Correspondence to: Uwe-G. Maier; email: maier@staff.uni- marburg.de J Mol Evol (2006) 62:674–681 DOI: 10.1007/s00239-005-0099-y