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Membrane‐specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803
Author(s) -
Spence Edward,
Sarcina Mary,
Ray Nicola,
Møller Simon Geir,
Mullineaux Conrad W.,
Robinson Colin
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03519.x
Subject(s) - periplasmic space , green fluorescent protein , biology , thylakoid , protein subcellular localization prediction , twin arginine translocation pathway , protein targeting , transport protein , synechocystis , microbiology and biotechnology , immunogold labelling , membrane protein , bacterial outer membrane , biochemistry , escherichia coli , chloroplast , mutant , membrane , gene , ultrastructure , anatomy
Summary The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec‐ and Tat‐type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat‐specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature‐size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA‐GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.