Model for Membrane Organization and Protein Sorting in the Cyanobacterium Synechocystis sp. PCC 6803 Inferred from Proteomics and Multivariate Sequence Analyses

Abstract

Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

“…NdbC was clearly located only in the PM, while no signal was detected from the thylakoid membrane fraction or from the soluble proteins (Fig. 5C), in line with results obtained by proteomic studies (Pisareva et al, 2011;Liberton et al, 2016).…”

“…Most of these transporters were reported previously to localize in the PM (Pisareva et al, 2011), in accordance with the location of the NdbC protein (Fig. 5C).…”

Assessment of the requirement for aquaporins in the thylakoid membrane of plant chloroplasts to sustain photosynthetic water oxidation

“…The Synechocystis protein has been shown to facilitate water transport across plasma membrane and to play a role in protection against hyperosmotic stress [24,25]. Thus far AqpZ has been found using proteomics in Synechocystis plasma membrane [26]. In the same report, proteomic data of Synechocystis thylakoid preparations have also been presented, but no aquaporins have been identified.…”

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…Cyanobacteria such as Synechocystis have two distinct inner membrane fractions: the cytoplasmic membrane (CM) and the thylakoid membrane (TM), be it that these two components may be partially connected (48,(55)(56)(57). Since our expression system did not include a specific membranetargeting signal, PR might localize in either one or in both of these membrane fractions.…”

“…To investigate this, we fractionated the membranes of a PR-His expressing Synechocystis strain, using sucrose-density gradient centrifugation and a two-phase separation system (46). Of the two membrane fractions obtained equal amounts of protein (in μg) were applied on SDS-PAGE, and analyzed using Western blots with three different antibodies: An anti-His-tag antibody to detect PRHis; an antibody against PsbC (CP43), a marker for the TM fraction (58); and an antibody against PixJ1, a marker specific for the CM fraction (47,48). Figure 2, show that the level of PR-His relative to the total amount of protein in the respective fraction is comparable for both fractions (panel A).…”