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Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria

Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria Plant Physiology, 2025, 199, kiaf237 https://doi.org/10.1093/plphys/kiaf237 Advance access publication 8 September 2025 News and Views Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria 1,2, Maneesh Lingwan * Plant Physiology, American Society of Plant Biologists Donald Danforth Plant Science Center, St. Louis, MO 63132, USA *Author for correspondence: [email protected] Cyanobacteria are oxygenic phototrophs that significantly con- contained factors implicated in metabolite transport, cell wall tribute to the carbon and nitrogen cycles by producing O and fix - maintenance, motility, and cell adhesion. Dual localization of 40 ing N . Unlike most prokaryotes and gram-negative bacteria, proteins was found in both the thylakoid lumen and P-OM pro- cyanobacteria possess a distinctive intracellular organization teomes, suggesting shared or transitional roles that might support that includes a plasma membrane (PM), a periplasm and outer TM biogenesis (Figure D). membrane with a peptidoglycan layer in between, thylakoid Proximity-based proteomics approaches can be utilized to membranes (TMs), and proteinaceous organelles like carboxy- understand how proteins are directed to their respective compart- somes, which support the carbon-concentrating mechanism ments. In cyanobacteria, proteins are translocated across mem- (Cameron et al. 2013; Dahlgren et al. 2021). These compartments branes either via the secretory (Sec) pathway to translocate contain unique proteins essential for various physiological func- unfolded proteins or the twin-arginine translocation (Tat) path- tions, such as photosynthesis, carbon fixation, and nutrient trans- way to translocate folded proteins (Frain et al. 2016). Proteins ex- port; however, the mechanisms for targeting proteins to these ported by the periplasm and the thylakoid lumen utilize locations have remained poorly understood. N-terminal signal sequences that guide their localization. These Proteomics involves assessing protein interactions, func- sequences are cleaved by different signal peptidases (SPI, SPII, or tions, compositions, structures, and their cellular activities. SPIII) that recognize distinct motifs. Most cyanobacteria have 1 Traditional proteomic analyses of cyanobacterial compartments set of genes for the Sec and Tat translocation systems, which have relied on biochemical fractionation and separation techni- are localized to the PM or TM (Russo and Zedler 2020), raising ques. These approaches have limitations regarding cross- questions about how proteins are specifically targeted to the contamination between fractions, resulting in uncertain cellular correct compartment. Dahlgren et al. (2025) suggested that signal localizations. To address these challenges, in this current issue sequences possess distinguishable characteristics that can recog- of Plant Physiology, Dahlgren et al. (2025) employed proximity- nize translocation complexes in the PM and TM. Signal sequence based proteomics techniques using ascorbate peroxidase analysis revealed a notable difference between proteins localized (APEX2) in the model cyanobacterium Synechococcus sp. PCC to the thylakoid lumen and those localized to the periplasm. In 7,002 to identify subcellular proteomes (Figure A). APEX2 cata- PCC 7,002, thylakoid lumen-targeted proteins translocated by lyzes a reaction using biotin-phenol and hydrogen peroxide as the Sec pathway typically possessed more hydrophobic and substrates, generating biotin-phenol radicals that covalently alpha-helical H-regions in their signal sequences compared to bind to proteins within a 20-nm radius from the site of production P-OM proteins. Interestingly, this trend of signal peptides was con- (Figure B). This method allows for specific labeling of membrane- served among other thylakoid-containing cyanobacteria, such as bound proteomes without requiring prior cell lysis, thus saving Synechocystis sp. PCC 6,803 and Nostoc sp. PCC 7,120, but not in time and minimizing cross-contamination and limitations (Rhee Gloeobacter violaceus PCC 7,421, a species that lacks internal TMs. et al. 2013). These conserved peptides indicate that the adaptations of the sig- Dahlgren et al. (2025) demonstrated that APEX2-based proteins nal sequence coevolved with the development of TM systems. were active in multiple cellular compartments, such as the cyto- APEX2 labeling-based proteomic analysis also revealed subdo- plasm, thylakoid lumen, and periplasm and outer membrane mains within major compartments. Distinct bait proteins present (P-OM). One important finding was the specific signal sequence as- within the same compartment, such as the cytoplasm or peri- signed to each subcellular compartment. For instance, APEX2 can plasm, were associated with overlapping yet distinct proteomes, be fused with extrinsic photosynthetic subunits of PSII such as lu- suggesting a degree of functional sub-organization. The relation- minal marker proteins PsbU and PsbQ, and A1097 and A1761 for ship between APEX2 labeling in the periplasmic and cytoplasmic labeling periplasmic marker proteins. Proteome enrichment anal- areas was stronger than the correlation between the cytoplasm ysis confirmed that the thylakoid lumen was enriched in proteins and thylakoid lumen. This variation is due to the Tat signal se- related to photosynthesis and the assembly of photosynthetic quences in periplasm-targeted bait proteins (A1097, A1761) facil- complexes, confirming its role in energy generation and oxygen- itating post-translational translocation of fully folded proteins evolving complex proteins (Figure C). In contrast, P-OM proteome into the periplasm. In contrast, Sec pathway substrates like Received May 1, 2025. Accepted May 18, 2025. © The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 | Plant Physiology, 2025, Vol. 199, No. 1 Figure. Proximity-based proteomics using APEX2 of cyanobacteria Synechococcus sp. PCC 7,002. A) Color-coded illustration showing the cellular and membrane structure of PCC 7002. The periplasmic space, which contains the cell wall, is surrounded by inner and outer cell membranes. TMs encase the thylakoid lumen. The zoom box depicts potential transient membrane connections between the inner and TMs. Hexagons within the cytoplasm represent carboxysomes. B) In the presence of biotin-phenol (BP) and hydrogen peroxide (H O ) substrate, APEX2 catalyzes a reaction creating a BP 2 2 radical. The BP radical covalently labels endogenous proteins. The functional classifications of proteins localized to C) thylakoid lumen, D) periplasm, and outer membrane proteome. thylakoid-targeted fusions (PsbQ) remain unfolded and minimize biology for engineering photosynthetic organisms with targeted cytoplasmic exposure and activity of APEX2. A total of 1,687 protein expression. proteins were identified, of which 271 were unlocalized. These un- Conflict of interest statement. None declared. localized proteins are possibly integral membrane proteins, for which the APEX2 labeling across the membrane poses challenges to precise localization. Data availability While APEX2 is highly suited for soluble proteins within No new data included in this article. membrane-bound compartments, it is less effective with inte- gral membrane proteins, probably due to the varying accessibil- ity of biotinylation sites. The study also identified a subset of 27 References dually localized proteins of unknown function, highlighting po- tential new players in membrane biogenesis and photosystem Cameron JC, Wilson SC, Bernstein SL, Kerfeld CA. Biogenesis of a assembly. Previous studies have limitations in identifying the bacterial organelle: the carboxysome assembly pathway. Cell. hydrophobic H-region trend, likely due to smaller datasets, 2013:155(5):1131–1140. https://doi.org/10.1016/j.cell.2013.10.044 lack of signal sequence type separation, and absence of thyla- Dahlgren KK, Ebmeier CC, Koke E, Cameron JC. Spatial proteomics koid lumen-specific proteomic data. Dahlgren et al. (2025) uti- reveals signal sequence characteristics correlated with localiza- lized a comprehensive dataset and improved methodology to tion in Cyanobacteria. Plant Physiol. 2025:198(4). https://doi.org/ provide a clearer picture of the signals guiding protein traffick - 10.1093/plphys/kiaf186 ing. The advantage of APEX2-based proximity proteomics has Dahlgren KK, Gates C, Lee T, Cameron JC. Proximity-based proteo- opened new avenues for understanding subcellular organiza- mics reveals the thylakoid lumen proteome in the cyanobacte- tion and quantitative profiling of the compartmentalome of cy- rium Synechococcus sp. PCC 7002. Photosynth Res. 2021:147(2): anobacteria. This approach can provide targets for synthetic 177–195. https://doi.org/10.1007/s11120-020-00806-y Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria | 3 Frain KM, Gangl D, Jones A, Zedler JAZ, Robinson C. Protein translo- cells via spatially restricted enzymatic tagging. Science. cation and thylakoid biogenesis in cyanobacteria. Biochim Biophys 2013:339(6125):1328–1331. https://doi.org/10.1126/science.123 Acta Bioenerg. 2016:1857(3):266–273. https://doi.org/10.1016/j. 0593 bbabio.2015.08.010 Russo DA, Zedler JAZ. Genomic insights into cyanobacterial protein Rhee H-W, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, translocation systems. Biol Chem. 2020:402(1):39–54. https://doi. Ting AY. Proteomic mapping of mitochondria in living org/10.1515/hsz-2020-0247 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Physiology Oxford University Press

Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria

Lingwan, Maneesh
Plant Physiology , Volume 199 (1): 1 – Sep 8, 2025
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Oxford University Press
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© The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists.
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0032-0889
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10.1093/plphys/kiaf237
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Abstract

Plant Physiology, 2025, 199, kiaf237 https://doi.org/10.1093/plphys/kiaf237 Advance access publication 8 September 2025 News and Views Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria 1,2, Maneesh Lingwan * Plant Physiology, American Society of Plant Biologists Donald Danforth Plant Science Center, St. Louis, MO 63132, USA *Author for correspondence: [email protected] Cyanobacteria are oxygenic phototrophs that significantly con- contained factors implicated in metabolite transport, cell wall tribute to the carbon and nitrogen cycles by producing O and fix - maintenance, motility, and cell adhesion. Dual localization of 40 ing N . Unlike most prokaryotes and gram-negative bacteria, proteins was found in both the thylakoid lumen and P-OM pro- cyanobacteria possess a distinctive intracellular organization teomes, suggesting shared or transitional roles that might support that includes a plasma membrane (PM), a periplasm and outer TM biogenesis (Figure D). membrane with a peptidoglycan layer in between, thylakoid Proximity-based proteomics approaches can be utilized to membranes (TMs), and proteinaceous organelles like carboxy- understand how proteins are directed to their respective compart- somes, which support the carbon-concentrating mechanism ments. In cyanobacteria, proteins are translocated across mem- (Cameron et al. 2013; Dahlgren et al. 2021). These compartments branes either via the secretory (Sec) pathway to translocate contain unique proteins essential for various physiological func- unfolded proteins or the twin-arginine translocation (Tat) path- tions, such as photosynthesis, carbon fixation, and nutrient trans- way to translocate folded proteins (Frain et al. 2016). Proteins ex- port; however, the mechanisms for targeting proteins to these ported by the periplasm and the thylakoid lumen utilize locations have remained poorly understood. N-terminal signal sequences that guide their localization. These Proteomics involves assessing protein interactions, func- sequences are cleaved by different signal peptidases (SPI, SPII, or tions, compositions, structures, and their cellular activities. SPIII) that recognize distinct motifs. Most cyanobacteria have 1 Traditional proteomic analyses of cyanobacterial compartments set of genes for the Sec and Tat translocation systems, which have relied on biochemical fractionation and separation techni- are localized to the PM or TM (Russo and Zedler 2020), raising ques. These approaches have limitations regarding cross- questions about how proteins are specifically targeted to the contamination between fractions, resulting in uncertain cellular correct compartment. Dahlgren et al. (2025) suggested that signal localizations. To address these challenges, in this current issue sequences possess distinguishable characteristics that can recog- of Plant Physiology, Dahlgren et al. (2025) employed proximity- nize translocation complexes in the PM and TM. Signal sequence based proteomics techniques using ascorbate peroxidase analysis revealed a notable difference between proteins localized (APEX2) in the model cyanobacterium Synechococcus sp. PCC to the thylakoid lumen and those localized to the periplasm. In 7,002 to identify subcellular proteomes (Figure A). APEX2 cata- PCC 7,002, thylakoid lumen-targeted proteins translocated by lyzes a reaction using biotin-phenol and hydrogen peroxide as the Sec pathway typically possessed more hydrophobic and substrates, generating biotin-phenol radicals that covalently alpha-helical H-regions in their signal sequences compared to bind to proteins within a 20-nm radius from the site of production P-OM proteins. Interestingly, this trend of signal peptides was con- (Figure B). This method allows for specific labeling of membrane- served among other thylakoid-containing cyanobacteria, such as bound proteomes without requiring prior cell lysis, thus saving Synechocystis sp. PCC 6,803 and Nostoc sp. PCC 7,120, but not in time and minimizing cross-contamination and limitations (Rhee Gloeobacter violaceus PCC 7,421, a species that lacks internal TMs. et al. 2013). These conserved peptides indicate that the adaptations of the sig- Dahlgren et al. (2025) demonstrated that APEX2-based proteins nal sequence coevolved with the development of TM systems. were active in multiple cellular compartments, such as the cyto- APEX2 labeling-based proteomic analysis also revealed subdo- plasm, thylakoid lumen, and periplasm and outer membrane mains within major compartments. Distinct bait proteins present (P-OM). One important finding was the specific signal sequence as- within the same compartment, such as the cytoplasm or peri- signed to each subcellular compartment. For instance, APEX2 can plasm, were associated with overlapping yet distinct proteomes, be fused with extrinsic photosynthetic subunits of PSII such as lu- suggesting a degree of functional sub-organization. The relation- minal marker proteins PsbU and PsbQ, and A1097 and A1761 for ship between APEX2 labeling in the periplasmic and cytoplasmic labeling periplasmic marker proteins. Proteome enrichment anal- areas was stronger than the correlation between the cytoplasm ysis confirmed that the thylakoid lumen was enriched in proteins and thylakoid lumen. This variation is due to the Tat signal se- related to photosynthesis and the assembly of photosynthetic quences in periplasm-targeted bait proteins (A1097, A1761) facil- complexes, confirming its role in energy generation and oxygen- itating post-translational translocation of fully folded proteins evolving complex proteins (Figure C). In contrast, P-OM proteome into the periplasm. In contrast, Sec pathway substrates like Received May 1, 2025. Accepted May 18, 2025. © The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 | Plant Physiology, 2025, Vol. 199, No. 1 Figure. Proximity-based proteomics using APEX2 of cyanobacteria Synechococcus sp. PCC 7,002. A) Color-coded illustration showing the cellular and membrane structure of PCC 7002. The periplasmic space, which contains the cell wall, is surrounded by inner and outer cell membranes. TMs encase the thylakoid lumen. The zoom box depicts potential transient membrane connections between the inner and TMs. Hexagons within the cytoplasm represent carboxysomes. B) In the presence of biotin-phenol (BP) and hydrogen peroxide (H O ) substrate, APEX2 catalyzes a reaction creating a BP 2 2 radical. The BP radical covalently labels endogenous proteins. The functional classifications of proteins localized to C) thylakoid lumen, D) periplasm, and outer membrane proteome. thylakoid-targeted fusions (PsbQ) remain unfolded and minimize biology for engineering photosynthetic organisms with targeted cytoplasmic exposure and activity of APEX2. A total of 1,687 protein expression. proteins were identified, of which 271 were unlocalized. These un- Conflict of interest statement. None declared. localized proteins are possibly integral membrane proteins, for which the APEX2 labeling across the membrane poses challenges to precise localization. Data availability While APEX2 is highly suited for soluble proteins within No new data included in this article. membrane-bound compartments, it is less effective with inte- gral membrane proteins, probably due to the varying accessibil- ity of biotinylation sites. The study also identified a subset of 27 References dually localized proteins of unknown function, highlighting po- tential new players in membrane biogenesis and photosystem Cameron JC, Wilson SC, Bernstein SL, Kerfeld CA. Biogenesis of a assembly. Previous studies have limitations in identifying the bacterial organelle: the carboxysome assembly pathway. Cell. hydrophobic H-region trend, likely due to smaller datasets, 2013:155(5):1131–1140. https://doi.org/10.1016/j.cell.2013.10.044 lack of signal sequence type separation, and absence of thyla- Dahlgren KK, Ebmeier CC, Koke E, Cameron JC. Spatial proteomics koid lumen-specific proteomic data. Dahlgren et al. (2025) uti- reveals signal sequence characteristics correlated with localiza- lized a comprehensive dataset and improved methodology to tion in Cyanobacteria. Plant Physiol. 2025:198(4). https://doi.org/ provide a clearer picture of the signals guiding protein traffick - 10.1093/plphys/kiaf186 ing. The advantage of APEX2-based proximity proteomics has Dahlgren KK, Gates C, Lee T, Cameron JC. Proximity-based proteo- opened new avenues for understanding subcellular organiza- mics reveals the thylakoid lumen proteome in the cyanobacte- tion and quantitative profiling of the compartmentalome of cy- rium Synechococcus sp. PCC 7002. Photosynth Res. 2021:147(2): anobacteria. This approach can provide targets for synthetic 177–195. https://doi.org/10.1007/s11120-020-00806-y Proximity-based proteomics reveals subcellular targeting sequence in Cyanobacteria | 3 Frain KM, Gangl D, Jones A, Zedler JAZ, Robinson C. Protein translo- cells via spatially restricted enzymatic tagging. Science. cation and thylakoid biogenesis in cyanobacteria. Biochim Biophys 2013:339(6125):1328–1331. https://doi.org/10.1126/science.123 Acta Bioenerg. 2016:1857(3):266–273. https://doi.org/10.1016/j. 0593 bbabio.2015.08.010 Russo DA, Zedler JAZ. Genomic insights into cyanobacterial protein Rhee H-W, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, translocation systems. Biol Chem. 2020:402(1):39–54. https://doi. Ting AY. Proteomic mapping of mitochondria in living org/10.1515/hsz-2020-0247

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Published: Sep 8, 2025

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