A model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803

Sammanfattning: Cyanobacteria constitute a unique group of eubacteria, possessing outer and plasma membranes as well as internal thylakoid membranes, the site for both photosynthesis and respiration. The combination of sucrose density centrifugation and two-phase partitioning methods leads to purification of different membranes from the cyanobacterium Synechocystis sp. PCC 6803 (referred to as Synechocystis). The standard proteomics methods, based on 1D- and 2D-gel electrophoresis, followed by protein digestion and MALDI-TOF MS, led to identification of 76 thylakoid and 51 plasma membrane proteins. In order to increase the number of identified proteins a shotgun proteomics approach was employed. Proteins were digested in complex mixtures, followed by LC separation of obtained peptides coupled to MS/MS. This approach led to identification of 379 different thylakoid and plasma membrane proteins, of which 124 were integral membrane proteins. The complex membrane organization of Synechocystis requires a unique system for transport and sorting of proteins into extracytosolic cell compartments. Obtained gel-based and shotgun proteomics data as well as results of the multivariate sequence analysis of both soluble and integral membrane proteins allowed to suggest a new mechanism for membrane organization and protein sorting in Synechocystis. The plasma and thylakoid membranes are proposed to be dynamically connected and both soluble and integral membrane proteins are inserted into connection points.The Synechocystis genome possesses two genes encoding leader peptidases. Proteomic studies revealed that Sll0716 is localized in the thylakoid membrane (LepT), whereas Slr1377 in the plasma membrane (LepP). The BN/SDS-PAGE of the total membranes from mutant LepT revealed that LepT is directly involved in the processing of several photosynthetic and lumenal subunits, required for the assembly and function of PSI and maintaining of the thylakoid membrane organization in Synechocystis.