Involvement of Ycf3, Ycf4 and BtpA in photosystem I biogenesis of Synechocystis sp. PCC6803.

Abstract

Cyanobacterial photosystem I serves as a model system to elucidate the biogenesis of a complex membrane protein on the molecular basis. PS I is an integral membrane complex, comprised of 11 protein subunits and a large number of cofactors. The proteins Ycf3, Ycf4 and BtpA have already been implicated as potential biogenesis-proteins on the basis of knock-out mutants, but their molecular function is unknown. In our group, the corresponding genes were cloned, overexpressed in E. coli and purified. Size exclusion chromatography shows homooligomerisation, with Ycf4 forming dimers at 40 kDa and Ycf3 and BtpA proteins existing in oligomeric complexes of 250 kDa and 650 kDa respectively. These data are supported by further experiments using the yeast-2-hybrid system and ligand-overlay assays. All proteins are associated with the thylakoid membrane of Synechocystis, as revealed by immunoelectronmicroscopy. Deletion mutants in Synechocystis sp. PCC6803 are fully segregated and show characteristic phenotypes. Cells lacking ycf3 posses no photosystem I and are unable to grow photoautotrophically. In contrast, ycf4 and btpA deletion strains grow in minimal media but show reduced levels of PS I. The degree of PS I reduction depends strongly on environmental factors as light and medium. Heterotrophically grown ycf4-deletion mutants express no PS I, however if the are grown photoautotrohically, PS I can be measured by low temperature fluorescence spectroscopy. Structure-function analysis of these deletion strains is under way and latest results will be presented during the meeting, as well as data on the composition of PS I in ycf4 and btpA deletion mutants. In addition, our deletion strains will be analysed by a proteomic approach. As an alternative to traditional 2-DE electrophoresis, we have established a system to identify integral and peripheral membrane proteins using a combined HPLC-electrophoresis technique and MALDI-TOF. Our data suggest a stabilizing or regulatory role for Ycf4 and BtpA, whereas Ycf3 is essential for the assembly of functional photosystem I.