Functional Proteomic Discovery of Slr0110 as a Central Regulator of Carbohydrate Metabolism in Synechocystis Species PCC6803

Abstract

The unicellular photosynthetic model-organism cyanobacterium Synechocystis sp. PCC6803 can grow photoautotrophically using CO 2 or heterotrophically using glucose as the sole carbon source. Several pathways are involved in carbon metabolism in Synechocystis, and the concerted regulation of these pathways by numerous known and unknown genes is critical for the survival and growth of the organism. Here, we report that a hypothetical protein encoded by the open reading frame slr0110 is necessary for heterotrophic growth of Synechocystis. The slr0110-deletion mutant is defective in glucose uptake, heterotrophic growth, and dark viability without detectable defects in autotrophic growth, whereas the level of photosystem II and the rate of oxygen evolution are increased in the mutant. Quantitative proteomic analysis revealed that several proteins in glycolysis and the oxidative pentose phosphate pathway are down-regulated, whereas proteins in photosystem II and phycobilisome are significantly up-regulated, in the mutant. Among the down-regulated proteins are glucose transporter, glucokinase, glucose-6-phosphate isomerase, and glucose-6-phosphate dehydrogenase and its assembly protein OpcA, suggesting that glycolysis, oxidative pentose phosphate, and glycogen synthesis pathways are significantly inhibited in the mutant, which was further confirmed by enzymatic assays and quantification of glycogen content. These findings establish Slr0110 as a novel central regulator of carbon metabolism in Synechocystis, and shed light on an intricate mechanism whereby photosynthesis and carbon metabolism are well concerted to survive the crisis when one or more pathways of the system are impaired. Molecular & Cellular

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs) *

“…2A). The identification of TM-containing proteins from soluble fraction is likely caused by inefficient precipitation of membranous vesicles from the soluble fraction, as previously discussed by us and others (11,28,30,31). If we include only two-or more-TM containing proteins, to exclude proteins with a signal peptide that were otherwise predicted by TMHMM as 1-TM containing, as the true TM-containing proteins, the number of uniquely identified TM-containing proteins can be reduced to 115 and 0 in the membrane and the soluble fractions, respectively, and 133 were from both fractions ( Fig.…”

“…Rational and Strategy-It has been repeatedly reported that large scale proteomic analyses for purified membranes identified many non-TM containing proteins that are known to be localized also in soluble fractions such as cytoplasm (11)(12)(13)(27)(28)(29), despite extensive and stringent purification steps being applied to ensure the purity of the isolated membranes. This observation suggested that many non-TM containing proteins have at least two subcellular pools, one pool associated with the membranes through either protein-protein interactions or protein-lipid interactions and the other was in the soluble space.…”

“…Cells were broken using a bead beater, and centrifuged with 5,000 ϫ g for 30 min at 4°C to remove glass beads and insoluble cell debris. The membrane and the soluble fractions were separated as previously described with slight modifications (11)(12)(13). Briefly, the whole cell lysate was centrifuged with 100,000 ϫ g at 4°C for 1 h. The pellet was collected as the membrane fraction and resuspended with the same cell lysis buffer.…”

“…1B. The whole cell lysate of Synechocystis was separated into the membrane and the soluble fractions (11)(12)(13). Proteins in each fraction were digested using the FASP approach (19), prefractionated with off-line basic RP-HPLC, and analyzed with high-resolution tandem MS coupled online to normal acidic nano-HPLC.…”

Cyanobacterial photo-driven mixotrophic metabolism and its advantages for biosynthesis

“…Low intensity blue light pulses can activate glucose metabolism and cell division without initiating photosynthesis [34]. Under heterotrophic conditions, certain membrane proteins of cyanobacteria can upregulate sugar utilization enzymes (e.g., fructose-1,6-bisphosphate aldolase and glucose transporter) and influence glucose uptake rate and dark viability [35]. Thirdly, cyanobacteria are able to close their TCA cycle by 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase [36].…”