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Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

ABSTRACT: We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify 97 cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. One quantification method was calibrated with artificial 15N-labelled proteins comprising concatenated proteotypic tryptic peptides and the other three methods were label-free (DDA-iBAQ, DDA-Top3, DIA-Top3) calibrated with the Sigma UPS2 standard protein mixture. As expected, the quantification methods gave individual distributions of abundance levels for each target protein that, for statistical validity, were not combined into single central tendency and variation metrics. Instead, overlapping distributions were merged to provide consensus abundance ranges. Quantification, expressed as copy numbers per cell (cpc), spanned four orders of magnitude, representing the complete abundance range from <1000 to >100,000 cpc of the proteins representing the complexes and pathways that were the focus of this study.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Synechocystis Sp. Pcc 6803 Substr. Gt-i

SUBMITTER: Philip Jackson

LAB HEAD: Neil Hunter

PROVIDER: PXD035632 | Pride | 2023-01-04

REPOSITORIES: Pride

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			Action	DRS
	LFQ-DDA_20180214_PJ_WL1-1.raw	Raw
	LFQ-DDA_20180214_PJ_WL1-2.raw	Raw
	LFQ-DDA_20180214_PJ_WL1-3.raw	Raw
	LFQ-DDA_20180214_PJ_WL2-1.raw	Raw
	LFQ-DDA_20180214_PJ_WL2-2.raw	Raw

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Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes. For transcription: Total RNA from top3-105 mutant and WT control cells after 8 hours at 36C in biological duplicates. For Top3-myc chromatin immunoprecipitation: DNA immunoprecipitated with mouse anti-Myc using chromatin extracts from cells expressing Top3-Myc from the endogenous locus at 30C in biological duplicates normalized to input DNA from wild type cells at 30C in biological duplicates. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from top3-105 mutant and wild type control cells after 8 hours at 36C in in biological duplicates normalized to input DNA from each strain.

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Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

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