title: Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AK, UK.
authors.forename: Edward
authors.initials: E
authors.lastname: Spence
authors.forename: Mary
authors.initials: M
authors.lastname: Sarcina
authors.forename: Nicola
authors.initials: N
authors.lastname: Ray
authors.forename: Simon Geir
authors.initials: SG
authors.lastname: Møller
authors.forename: Conrad W
authors.initials: CW
authors.lastname: Mullineaux
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase