MBE Advance Access published online on July 3, 2008
Molecular Biology and Evolution, doi:10.1093/molbev/msn147
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Research Article |
Nucleus-encoded periplastid-targeted EFL in chlorarachniophytes
Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada
* Corresponding author: email: pkeeling{at}interchange.ubc.ca, phone: 604-822-4906, fax: 604-822-6089
Received for publication April 17, 2008. Revision received June 25, 2008. Accepted for publication June 26, 2008.
Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins, since none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from seven chlorarachniophytes and two distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins, and which we conclude is most likely PPC-targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with post-translational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein, but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor-1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all three classes of targeted protein by comparing all examples of each class from expressed sequence tag (EST) surveys of B. natans and G. stellata. No recognizable difference between plastid- and PPC-targeted proteins was observed, but nucleomorph-encoded transit peptides differ, likely reflecting high AT content of nucleomorph genomes. Taken together, the data suggest that the system that directs proteins to the PPC in chlorarachniophytes uses a bipartite targeting sequence, as does the PPC-targeting system that evolved independently in cryptomonads.
Key Words: protein targeting • plastids • chlorarachniophytes • transit peptides • Gymnochlora stellata
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  Y. Hirakawa, K. Nagamune, and K.-i. Ishida Protein targeting into secondary plastids of chlorarachniophytes PNAS, August 4, 2009; 106(31): 12820 - 12825. [Abstract] [Full Text] [PDF]
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