Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.

Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle, tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites, the elongation phase of protein synthesis is regulated by the Toxoplasma oxygen-sensing protein, PHYb. PHYb acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PHYb, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2 . In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions, CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular stress-induced eEF2 kinase. IMPORTANCE Although it is an obligate intracellular parasite, Toxoplasma must be able to survive in the extracellular environment. Our previous work indicated that ensuring that elongation continues during protein synthesis is part of this stress response and that this is due to preventing phosphorylation of elongation factor 2. But the identity of the eEF2 kinase has remained unknown in Toxoplasma and other protozoan parasites. Here, we identify CDPK3 as the first protozoan eEF2 kinase and demonstrate that it is part of a stress response initiated when parasites are exposed to extracellular stress. We also demonstrate that CDPK3 engages eEF2 as a result of its relocalization from the plasma membrane to the cytosol.

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20Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined 21 different features of cyanobacteria to learn about the structure-function relationships between the 22 of the cell that has remained challenging to study with existing methods, using a PsbU-APEX2 32 gene fusion. This study demonstrates the power of APEX2 as a tool to study the cell biology of 33 intracellular features of cyanobacteria with enhanced spatiotemporal resolution. 34 35 Keywords: APEX2, proximity-based proteomics, thylakoid lumen, sub-cellular localization, 36 cyanobacteria, photosynthesis, Photosystem II 37 the reaction, cells were lysed by bead beating and a streptavidin blot confirmed the ability of 103 APEX2 to biotinylate proteins in PCC 7002 ( Figure 1C). Biotin labeling only occurred in the 104 presence of APEX2, BP, and H2O2, demonstrating reaction specificity in vivo. Furthermore, the 105 rapid reaction enables precise temporal control of labeling. 106 107 108 109 Figure 2. Enrichment of proteins biotinylated by cytoplasmic APEX2 in vivo 110Cells expressing GFP or GFP-APEX2 were incubated with BP and exposed to H2O2. Biotinylated 111 proteins were captured from cell lysates on streptavidin coated magnetic beads. Fractions from 112 each enrichment step were separated by SDS-PAGE and then silver stained for contrast or 113 transferred to a nitrocellulose membrane and probed with specific antibodies. (A) Silver stain of 114 noted fractions from unlabeled (GFP) or labeled (GFP-APEX2) lysates. (B) Biotinylated proteins 115 are only detected in fractions containing APEX2 and are enriched on streptavidin beads. (C) 116Expected self-labeling (biotinylation) of GFP-APEX2 (54 kDa, marked with *) is confirmed by 117 immunoblotting against GFP. (D) RbcL (55 kDa), a cytoplasmic protein expected to be labeled by 118 GFP-APEX2 was specifically captured on beads incubated with GFP-APEX2. 119