Recombinant Prochlorococcus marinus subsp. pastoris Photosystem I assembly protein ycf3 (ycf3)
Description
Fundamental Characteristics of Ycf3
Ycf3 is a highly conserved protein encoded in the plastid genomes of photosynthetic eukaryotes and cyanobacteria. In cyanobacteria like Prochlorococcus marinus, it functions as an essential assembly factor for the photosystem I complex. The importance of Ycf3 is evidenced by its conservation across diverse photosynthetic organisms, suggesting a critical and evolutionarily preserved role in photosynthesis .
The ycf3 gene is typically part of a polycistronic transcriptional unit, often found in a gene cluster that includes rps9, ycf4, and rps18, as demonstrated in studies of chloroplast gene organization. Northern blot analyses have shown that this cluster is transcribed into a large RNA of approximately 8.0 kb, with additional processing resulting in smaller transcripts . This genomic organization highlights the coordinated expression of genes involved in photosynthetic apparatus assembly.
Prochlorococcus marinus, the source organism for the recombinant Ycf3 protein, belongs to a genus of extremely abundant marine cyanobacteria that contribute significantly to global photosynthesis. The subspecies pastoris represents one of several ecotypes adapted to specific ocean light and nutrient conditions .
Tetratrico-Peptide Repeat Domains
The Ycf3 protein contains three tetratrico-peptide repeats (TPRs), which are structural motifs known to function as sites for protein-protein interactions. These TPR domains are crucial for Ycf3's function as an assembly chaperone . The precise arrangement of these domains enables Ycf3 to interact specifically with photosystem I subunits during the assembly process.
Mutations in these TPR domains have significant consequences for PSI assembly and organism viability. Research has shown that mutations Y95A/Y96A and Y142A/W143A in the second and third TPR repeats lead to a modest decrease in PSI accumulation but prevent photoautotrophic growth and cause enhanced light sensitivity, despite the accumulated PSI complex remaining functionally intact . This phenotype can be reversed under anaerobic conditions, suggesting it results from photooxidative damage rather than direct inhibition of photosynthesis.
Assembly Chaperone Function
Ycf3 serves as a critical assembly chaperone for the PSI complex. Temperature-shift experiments using a temperature-sensitive ycf3 mutant have conclusively demonstrated that Ycf3 is required specifically for PSI assembly but not for the stability of already assembled PSI complexes . This finding indicates that Ycf3 functions transiently during the assembly process rather than as a permanent structural component of PSI.
Immunoprecipitation experiments have shown that Ycf3 directly interacts with at least two PSI subunits: PsaA (a core reaction center protein) and PsaD (a peripheral subunit on the stromal side) . These specific interactions suggest that Ycf3 helps coordinate the incorporation of these subunits during PSI assembly.
Cooperative Assembly Mechanisms
The assembly of PSI requires the coordinated action of multiple factors working in concert. Recent research has revealed that Ycf3 works as part of a dedicated PSI assembly apparatus. This apparatus consists of two primary modules:
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The Ycf3-Y3IP1 module: Facilitates the assembly of reaction center subunits
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The Ycf4 module: Mediates the integration of peripheral PSI subunits and light-harvesting complexes (LHCIs) into the PSI reaction center subcomplex
This modular assembly system demonstrates the sophisticated molecular machinery required for constructing the large and complex PSI structure. The Ycf3-containing module appears to function earlier in the assembly process, preparing the core components before the integration of peripheral elements .
Localization and Membrane Association
Ycf3 is loosely attached to the stromal side of the thylakoid membrane . This localization is consistent with its role in assembling components on the reducing side of PSI. Fractionation studies using sucrose density gradient centrifugation (SDGC) have shown that Ycf3 predominantly appears in upper gradient fractions, indicating its presence as either free protein or in small protein complexes rather than being permanently integrated into large membrane complexes .
Direct Interaction Partners
Extensive research using various protein-protein interaction methods has identified several direct interaction partners of Ycf3. These interactions are crucial for understanding the mechanistic details of PSI assembly. Table 1 summarizes the confirmed interaction partners of Ycf3 and their roles in PSI assembly.
Table 1: Confirmed Interaction Partners of Ycf3
The interaction with Y3IP1 (Ycf3-Interacting Protein 1) is particularly significant in eukaryotic photosynthetic organisms. Y3IP1 is a nucleus-encoded thylakoid protein that cooperates with Ycf3 in PSI assembly . Interestingly, Ycf51, which is found in cyanobacteria, appears to serve a similar function to Y3IP1 in eukaryotes, despite lacking sequence similarity .
Assembly Factor Interactions
The interaction between Ycf3 and Ycf51 (in cyanobacteria) has been characterized in detail. Cross-linking mass spectrometry identified 16 intermolecular cross-links between these proteins, confirming their physical association . Molecular docking analyses further revealed complementary charged surfaces that facilitate this interaction.
The different but overlapping distributions of Ycf3 and Ycf51 in gradient fractions of thylakoid proteins suggest that Ycf3 is involved in earlier steps of PSI assembly than Ycf51 . This temporal separation of function helps coordinate the complex assembly process of PSI.
Gene Disruption Studies
Much of our understanding of Ycf3 function comes from gene disruption studies. In these experiments, the ycf3 gene is inactivated, resulting in the inability to accumulate functional PSI complexes . These mutants are unable to grow photoautotrophically, demonstrating the essential nature of Ycf3 for photosynthesis.
Temperature-sensitive mutations have been particularly valuable for studying the role of Ycf3. By shifting to restrictive temperatures, researchers can observe the immediate effects of Ycf3 inactivation, which revealed that new PSI assembly is halted while existing PSI complexes remain stable .
Recombinant Production and Applications
The recombinant Prochlorococcus marinus subsp. pastoris Photosystem I assembly protein Ycf3 is commercially available from suppliers such as MyBioSource.com at approximately $690.00 (as of February 2025) . This recombinant protein serves as a valuable tool for researchers investigating:
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The specific mechanisms of PSI assembly
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Protein-protein interactions involved in photosynthetic complex formation
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Evolutionary aspects of photosynthesis across different organisms
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Development of enhanced photosynthetic systems for biotechnological applications
Heterologous expression systems, such as Escherichia coli, have been successfully employed to produce recombinant Ycf3 for in vitro studies . These systems allow for the addition of affinity tags (such as GST or His tags) that facilitate purification and interaction studies.
Evolutionary Conservation and Comparative Analysis
The Ycf3 protein sequence is highly conserved across cyanobacteria, algae, and plants, highlighting its fundamental importance in oxygenic photosynthesis . This conservation extends to the functional level, as Ycf3 proteins from diverse organisms perform similar roles in PSI assembly.
Functional Analogs Across Species
While Ycf3 is conserved across photosynthetic organisms, its partner proteins have diverged. In cyanobacteria like Prochlorococcus, Ycf3 interacts with Ycf51, whereas in eukaryotic photosynthetic organisms (algae and plants), it partners with Y3IP1 . Despite the lack of sequence similarity between Ycf51 and Y3IP1, both appear to perform analogous functions in their respective organisms, suggesting convergent evolution of PSI assembly mechanisms.
Interestingly, heterologous complementation experiments have shown that Y3IP1 from Arabidopsis cannot functionally complement a ycf51 deficiency in cyanobacteria . This indicates that these proteins have co-evolved with their respective interaction partners in ways that prevent cross-species functionality.
Current Research and Future Directions
The study of recombinant Prochlorococcus marinus Ycf3 continues to advance our understanding of photosynthesis. Current research focuses on:
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Determining the precise molecular mechanisms by which Ycf3 facilitates PSI subunit assembly
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Identifying additional components of the PSI assembly apparatus
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Exploring potential applications in improving photosynthetic efficiency
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Understanding how Ycf3 function may differ across various ecological strains of Prochlorococcus
Challenges and Opportunities
Despite significant progress, several challenges remain in fully understanding Ycf3 function. The transient nature of its interactions during assembly makes them difficult to capture and characterize. Advanced techniques such as time-resolved cryo-electron microscopy and in vivo labeling approaches may help overcome these limitations.
The availability of recombinant Prochlorococcus Ycf3 provides opportunities for detailed structural studies that could reveal the precise molecular mechanisms of its chaperone function. Such insights could potentially be applied to enhance photosynthetic efficiency in both natural and artificial systems.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If a specific tag type is required, please inform us, and we will prioritize its development.
Target Background
KEGG: pmm:PMM0132
STRING: 59919.PMM0132
Q&A
What is the Ycf3 protein and what is its primary function?
Ycf3 is an essential protein for the assembly of the Photosystem I (PSI) complex that acts at a post-translational level. Research has conclusively demonstrated that Ycf3 functions as a molecular chaperone, facilitating the proper assembly of PSI by interacting directly with specific PSI subunits, particularly PsaA and PsaD . Knockout studies have shown that in the absence of Ycf3, organisms fail to accumulate functional PSI complexes, rendering them incapable of photoautotrophic growth . This protein represents a critical component of the photosynthetic machinery, with its primary function being to ensure the correct folding and assembly of the PSI complex components.
How is Ycf3 localized within the cell?
Ycf3 is an extrinsic protein associated with the thylakoid membranes . Though not an integral membrane protein itself, it interacts closely with membrane-bound proteins and complexes. Immunoblot analyses of thylakoid membranes separated by two-dimensional gel electrophoresis have shown that Ycf3 specifically interacts with PSI subunits but not with components from other photosynthetic complexes . This localization pattern is consistent with its role in PSI assembly, allowing it to interact with newly synthesized PSI components as they are integrated into the thylakoid membrane system.
What techniques can be used to study Ycf3-protein interactions?
Several experimental approaches have proven effective for studying Ycf3 interactions:
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Epitope tagging and affinity purification: Adding a FLAG or HA tag to the C-terminus of Ycf3 allows for immunoprecipitation of the protein complex without disrupting function . This technique has successfully identified proteins that interact with Ycf3.
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Two-dimensional gel electrophoresis: This method has been used to separate thylakoid membrane components and identify Ycf3 interactions through immunoblot analysis .
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Immunoprecipitation: Direct evidence of Ycf3's interaction with PSI subunits PsaA and PsaD has been obtained through immunoprecipitation experiments .
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Sucrose density gradient ultracentrifugation: This technique has been employed to fractionate Ycf3-containing complexes and determine their composition and approximate molecular weight .
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LC-MS/MS analysis: This method has been used to identify peptide fragments from Ycf3-interacting proteins in purified preparations .
How can researchers generate and characterize Ycf3 mutants?
Generation of Ycf3 mutants has been accomplished through several approaches:
Site-directed mutagenesis: Specific mutations in the TPR domains (Y95A/Y96A and Y142A/W143A) have been created to study the functional importance of these regions . These mutations target conserved residues within the protein-protein interaction domains.
Random mutagenesis: Degenerate oligonucleotides have been used to introduce mutations in the conserved N-terminal region upstream of the TPR domains . This approach has yielded temperature-sensitive mutants that are particularly valuable for studying protein function under controlled conditions.
Characterization methods include:
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Temperature-shift experiments: Using temperature-sensitive mutants to study the role of Ycf3 in assembly versus stability of PSI .
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Analysis of photosynthetic growth: Assessment of photoautotrophic growth capability under various light intensities to determine functional impairment .
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Immunoblot analysis: Quantification of PSI subunit accumulation to measure the impact of mutations on complex assembly .
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Measurement of PSI activity: Functional assays to determine if the assembled PSI complexes maintain electron transfer capability .
What considerations are important when creating tagged versions of Ycf3?
When creating tagged versions of Ycf3 for detection and purification, several important considerations must be addressed:
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Tag placement: The C-terminus of Ycf3 displays considerable interspecific sequence variation and has proven to be a suitable site for epitope tag addition without disrupting function . This region tolerates modification because it is less conserved across species.
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Verification of functionality: Proper validation requires demonstrating that the tagged version can complement a Ycf3 knockout. Plants expressing Ycf3-FLAG have been shown to grow photoautotrophically and accumulate normal levels of PSI, confirming that the tagged protein retains full functionality .
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Control experiments: It's essential to compare tagged strains with both wild-type and knockout strains to verify that the tag doesn't alter protein function. This includes assessment of growth, pigmentation, and PSI accumulation .
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Expression system: When studying Prochlorococcus specifically, consideration must be given to codon optimization for this organism's unique genomic properties.
What is the precise mechanism by which Ycf3 facilitates PSI assembly?
The molecular mechanism of Ycf3-mediated PSI assembly involves a sequential process of protein-protein interactions:
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Initial binding to PSI reaction center subunits: The Ycf3-Y3IP1 module transiently binds to newly synthesized reaction center (RC) subunits, specifically PsaA and PsaB . This represents an essential initial step in PSI assembly.
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Chaperone function: Ycf3 acts as a chaperone that facilitates the proper folding and interaction of PSI subunits. Its three TPR domains provide multiple sites for protein-protein interactions that help position subunits correctly for assembly .
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Cooperative action with other assembly factors: Ycf3 works in concert with other factors, particularly Y3IP1 and Ycf4. While Ycf3-Y3IP1 complex is involved in the initial assembly steps, Ycf4 appears to bind to the PSI RC subcomplex formed through Ycf3 assistance .
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Transient interactions: The interaction between the Ycf3-Y3IP1 complex and PSI subunits appears to be unstable and transient, as demonstrated by sucrose density gradient ultracentrifugation experiments. This suggests Ycf3 functions catalytically rather than as a permanent structural component .
How does the Ycf3-Y3IP1 complex function in PSI assembly?
The Ycf3-Y3IP1 complex plays a critical role in the early stages of PSI assembly:
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Complex formation: Y3IP1 (also known as CGL59 in Chlamydomonas) forms a stable complex with Ycf3 . This interaction has been confirmed through affinity purification of both Ycf3-HA and Y3IP1-HA tagged proteins.
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PSI reaction center assembly: The complex is specifically involved in an initial step of PSI reaction center assembly, as demonstrated by its transient binding to newly synthesized RC subunits .
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Mutual stability dependency: Y3IP1 appears important for the accumulation of Ycf3. In a Y3IP1 knockout mutant (ΔY3IP1), Ycf3 accumulated to only ~30% of control levels, while PSI accumulated to merely ~5% . This suggests the proteins stabilize each other.
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Functional relationship: While both proteins are essential for PSI assembly, they appear to have distinct but complementary roles. Experimental data indicates that:
| Protein | Effect of Knockout | PSI Accumulation | Direct Interactions |
|---|---|---|---|
| Ycf3 | Lethal | None | PsaA, PsaD |
| Y3IP1 | Lethal | ~5% of normal | Ycf3, Ycf4 |
What is the relationship between light intensity and Ycf3 function?
The function of Ycf3 appears to be particularly sensitive to light conditions:
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Light sensitivity of mutants: Mutations in the TPR domains (Y95A/Y96A and Y142A/W143A) cause enhanced light sensitivity even when PSI accumulates to levels that should support photosynthetic growth . This phenotype suggests that improperly assembled PSI may be particularly susceptible to photooxidative damage.
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Anaerobic rescue: The light-sensitive phenotype of these mutants can be reversed under anaerobic conditions, confirming that photooxidative damage is the primary cause of the growth defect rather than a direct failure of PSI function .
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Reactive oxygen species connection: Research suggests a link between light intensity, intracellular reactive oxygen species concentration, and protein synthesis rates . This connection may explain why higher light intensities exacerbate the phenotypes of Ycf3 mutants.
How does Ycf3 function differ between cyanobacteria, algae, and higher plants?
While Ycf3 is conserved across photosynthetic organisms, there are noteworthy differences in its function:
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Conservation level: The sequence of Ycf3 is conserved in cyanobacteria, algae, and plants, particularly in the N-terminal region and TPR domains . This conservation suggests the fundamental mechanism of action is maintained across evolutionary lines.
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Expression and regulation: In higher plants, Ycf3 is encoded in the plastid genome, while associated factors like Y3IP1 are nuclear-encoded . This dual genetic control allows for coordinated regulation of photosystem assembly.
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Interaction partners: While the core function of Ycf3 in PSI assembly is conserved, the specific interaction partners and assembly intermediates may vary between organisms. For instance, the roles of Y3IP1 and Ycf4 appear to have some organism-specific features .
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Phenotypic consequences: Complete loss of Ycf3 function is lethal in photoautotrophic conditions across studied organisms due to the absence of PSI, but the severity of partial loss-of-function may vary between species .
What evolutionary insights can be gained from studying Ycf3?
Several evolutionary insights emerge from Ycf3 research:
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Conserved assembly machinery: The conservation of Ycf3 across diverse photosynthetic organisms indicates that the fundamental mechanism of PSI assembly was established early in the evolution of photosynthesis and has been maintained.
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Co-evolution of interaction partners: The coordinated action of Ycf3 with other assembly factors suggests co-evolution of these proteins to maintain efficient PSI assembly throughout evolutionary divergence of photosynthetic organisms.
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Organellar genome retention: The fact that Ycf3 remains encoded in the plastid genome of plants, despite extensive transfer of chloroplast genes to the nucleus during evolution, suggests there may be advantages to local expression of this assembly factor near its site of action.
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Role in adaptation: The sensitivity of Ycf3 function to light conditions may reflect its role in adaptation to varying light environments, a critical factor in the evolutionary success of photosynthetic organisms.
What are the primary technical challenges in studying Ycf3?
Researchers face several technical challenges when studying Ycf3:
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Detection difficulties: Early studies noted difficulties in detecting the native Ycf3 protein using specific antibodies . This necessitated the development of epitope-tagged versions for reliable detection.
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Transient interactions: The transient nature of Ycf3's interactions with PSI subunits during assembly makes capturing these interactions challenging. Sucrose density gradient experiments show that most Ycf3-HA and Y3IP1 separate from PSI subunits during analysis .
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Low abundance: The relatively low abundance of Ycf3 and assembly intermediates in the cell makes biochemical characterization difficult. As noted in one study, "the amount of PSI RC subunits present in the Ycf3–Y3IP1 module was very low, it was difficult to show the presence of cofactors in the preparation" .
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Coordination with cofactor insertion: Understanding how Ycf3-mediated protein assembly coordinates with the insertion of essential cofactors (like iron-sulfur clusters) remains technically challenging to observe and analyze.
What future research directions could advance our understanding of Ycf3?
Several promising research directions could enhance our understanding of Ycf3:
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Large-scale biochemical preparation: As noted in one study, "it is important to make a large-scale preparation to allow for detailed biochemical characterization as a future study" . This would enable more comprehensive analysis of the composition and structure of Ycf3-containing complexes.
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Structural studies: Determining the three-dimensional structure of Ycf3, particularly in complex with interaction partners, would provide invaluable insights into its mechanism of action.
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Time-resolved analysis of assembly: Developing methods for capturing and analyzing the sequential steps of PSI assembly in real-time would help clarify the precise role of Ycf3 at each stage.
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Comparative analysis across species: More systematic comparison of Ycf3 function across diverse photosynthetic organisms could reveal adaptations specific to particular environmental niches.
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Investigation of regulatory mechanisms: Understanding how Ycf3 activity is regulated in response to environmental conditions and developmental stages would provide a more comprehensive picture of its role in photosynthetic organisms.
