Use of aminoglycoside adenyltransferase translational fusions to determine topology of thylakoid membrane proteins

We have developed a system to examine the topology of thylakoid membrane proteins using the bacterial aadA gene as a reporter. Translational fusions that place the aminoglycoside adenyltransferase domain in the stroma should provide high antibiotic resistance, while those that place it in the thylakoid lumen should give rise to low resistance. Genes encoding chimeric polypeptides consisting of AadA fused to varying lengths of the PsaA polypeptide, whose topology is known, were introduced into the chloroplast genome of Chlamydomonas reinhardtii. As expected, chimeras with an even number of α-helices in general resulted in higher resistance. This effect was not due to differences in expression or in catalytic activity. This system should prove useful in analysis of novel proteins predicted to be localized to the thylakoid membrane.