Recombinant Agrobacterium tumefaciens Membrane protein insertase YidC (yidC)
Description
Introduction to YidC in Bacterial Membrane Biogenesis
YidC is a critical membrane protein insertase belonging to the conserved Oxa1 superfamily, essential for the biogenesis of bacterial inner membranes. It facilitates the insertion, folding, and assembly of membrane proteins, either independently or in concert with the Sec translocon . While extensively studied in Escherichia coli, YidC homologs in Agrobacterium tumefaciens remain less characterized. Recombinant YidC from A. tumefaciens is engineered to study its structural and functional roles in membrane protein biogenesis, particularly in the context of plant pathogenicity and virulence mechanisms .
Recombinant YidC Production in Agrobacterium tumefaciens
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Cloning: Full-length yidC (1–620 amino acids) fused to an N-terminal His-tag for affinity purification .
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Expression: Optimized in E. coli under induction with arabinose or IPTG .
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Purification: Detergent-solubilized membranes followed by nickel-affinity chromatography .
Table 1: Key Properties of Recombinant Agrobacterium vitis YidC (Homolog)
Substrate Recognition and Insertion
YidC interacts with nascent membrane proteins via its hydrophilic groove, reducing the energy barrier for TM domain insertion into the lipid bilayer . For example:
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Pf3 coat protein: Insertion efficiency increases 1.5–1.8-fold in YidC-enriched membranes .
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SecG: Dual-orientation insertion is enhanced by YidC, as shown by protease-protected fragments .
Interaction Partners
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YibN: A newly identified interactor in E. coli stimulates YidC’s insertase activity and lipid scramblase function .
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SecYEG: Forms transient complexes for co-translational insertion of Sec-dependent substrates .
In Vitro Insertion Assays
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Inverted membrane vesicles (INVs): YidC-enriched INVs enhance insertion of Pf3, M13, and F0c by ~1.5–1.8-fold .
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Protease protection assays: Validate membrane integration by detecting protected fragments (e.g., SecG MPF1/2) .
Complementation Studies
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YidC-depletion strains: Fail to assemble functional F0F1 ATPase, confirming YidC’s role in energy transduction .
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Alanine mutagenesis: Residues T362 (TM2) and Y517 (TM6) are critical for YidC stability and activity .
Challenges and Future Directions
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order. We will prepare according to your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please notify us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
The tag type will be decided during the production process. If you have a specific tag type requirement, please inform us, and we will prioritize developing the specified tag.
Target Background
KEGG: atu:Atu0384
STRING: 176299.Atu0384
Q&A
What is YidC and what is its fundamental role in bacterial membrane biology?
YidC is a prominent member of the Oxa1 superfamily that plays an essential role in bacterial inner membrane biogenesis. It significantly influences membrane protein composition and lipid organization within the bacterial cell . In the context of A. tumefaciens research, YidC functions both in conjunction with the Sec translocon and independently as:
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A membrane protein insertase facilitating proper folding of multi-pass membrane proteins
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A lipid scramblase contributing to bilayer organization
The dual functionality of YidC makes it particularly interesting for studying membrane protein biogenesis in A. tumefaciens, a bacterium with both circular and linear chromosomes that has been extensively studied for its genetic transformation capabilities .
How does YidC interact with other membrane components in bacterial systems?
Recent research using proximity-dependent biotin labeling (BioID) has identified YibN as a crucial component within the YidC protein environment . This interaction was confirmed through multiple experimental approaches:
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Affinity purification-mass spectrometry assays conducted on native membranes
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On-gel binding assays with purified proteins
These findings suggest that YidC does not function in isolation but operates within a network of protein interactions that collectively ensure proper membrane protein insertion and organization.
What substrates are known to be processed by YidC in bacterial systems?
YidC has been demonstrated to facilitate the insertion of several key membrane proteins, including:
These findings suggest that YidC may serve as a critical insertase for numerous membrane proteins in A. tumefaciens, potentially including those involved in its unique DNA transfer capabilities.
What experimental approaches are most effective for studying YidC-dependent insertase activity?
Based on current research methodologies, a multi-faceted approach is recommended:
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Proximity-based labeling techniques: The BioID system (using BirA R118G mutant biotin ligase) fused to YidC has proven effective for identifying potential interacting partners .
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Membrane isolation and protein extraction protocol:
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Interactome analysis workflow:
This comprehensive approach allows for the identification of both direct and indirect interactors of YidC, providing insight into its functional network.
How can researchers assess the impact of YidC on membrane protein insertion efficiency?
To quantitatively evaluate YidC's contribution to membrane protein insertion, researchers should employ:
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Co-expression assays: Express YidC alongside known substrate proteins (such as phage coat proteins or ATP synthase subunit c) and measure incorporation rates .
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In vitro reconstitution systems: Purify YidC and test its insertase activity using artificial membrane systems with fluorescently labeled substrate proteins.
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Comparative genomics approach: Leverage A. tumefaciens' close relationship to Rhizobium bacteria to identify conserved and divergent aspects of YidC function across related species.
What are the challenges in expressing and purifying functional recombinant A. tumefaciens YidC?
Several technical challenges must be addressed:
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Membrane protein solubilization: Optimization of detergent type and concentration is critical; research suggests 1% DDM is effective for YidC extraction while maintaining protein-protein interactions .
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Expression system selection: Consider using specialized expression systems designed for membrane proteins, potentially leveraging A. tumefaciens' own genetic machinery given its unique chromosomal structure (both circular and linear chromosomes) .
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Functional validation: Confirm that recombinant YidC maintains insertase activity through in vitro assays with known substrates such as ATP synthase subunit c .
How does the YidC-YibN interaction influence membrane biology in bacterial systems?
The recently identified interaction between YidC and YibN has significant implications for membrane biology:
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Enhanced substrate processing: YibN has been shown to enhance the production and membrane insertion of YidC substrates, including M13 and Pf3 phage coat proteins, ATP synthase subunit c, and small membrane proteins like SecG .
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Membrane lipid dynamics: Overproduction of YibN stimulates membrane lipid production and promotes inner membrane proliferation, potentially by interfering with YidC's lipid scramblase activity .
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Functional consequences: The data suggests that YibN serves as both a physical and functional interactor of YidC, with direct implications for membrane protein insertion and lipid organization .
This interaction may be particularly relevant in A. tumefaciens given its specialized membrane requirements for host infection and DNA transfer processes.
What is the relationship between genome maintenance factors and YidC function in A. tumefaciens?
Research on A. tumefaciens has revealed important connections between genome maintenance and membrane biology:
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CcrM DNA methyltransferase: Depletion of CcrM in A. tumefaciens results in slow growth, particularly in complex media, with cells showing elongation and morphological abnormalities before eventual lysis .
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Growth condition dependencies: While CcrM appears dispensable in minimal media, it becomes essential for A. tumefaciens survival in complex media .
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Genomic methylation patterns: CcrM methylates GANTC motifs in the A. tumefaciens genome, with varying methylation levels observed across different genomic regions .
These findings suggest potential connections between genome maintenance, methylation patterns, and membrane protein expression (including YidC) that warrant further investigation in A. tumefaciens.
How might the unique genetic transfer capabilities of A. tumefaciens affect YidC expression and function?
A. tumefaciens is renowned for its ability to transfer DNA between kingdoms through its Ti plasmid system . This unique capability raises important questions about YidC:
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Host-pathogen interface: YidC may play a role in the assembly of membrane components required for A. tumefaciens attachment to plant cells and subsequent DNA transfer .
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Ti plasmid effects: The presence and activation of the Ti plasmid may influence YidC expression patterns, potentially through regulatory mechanisms connected to the virulence (Vir) genes .
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Experimental considerations: When using A. tumefaciens as a tool for plant transformation, researchers should consider how modifications to improve transformation efficiency might impact membrane protein insertion systems including YidC.
What growth conditions optimize recombinant YidC expression in A. tumefaciens?
Optimal cultivation conditions for A. tumefaciens must balance growth and protein expression:
When designing expression systems, consider that A. tumefaciens shows different growth behaviors and morphological characteristics depending on media composition, which may impact membrane protein expression and insertion .
How can researchers leverage A. tumefaciens' natural genetic engineering capabilities when studying YidC?
A. tumefaciens' natural capacity to transfer DNA can be repurposed for YidC research:
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Binary vector systems: Modified Ti plasmids have been engineered to create binary and co-integrative vector systems , which could be adapted to study YidC variants in both bacterial and plant cells.
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Host range considerations: Different wildtype A. tumefaciens strains show varying transformation efficiencies with different plant species . Researchers should select appropriate strains based on experimental needs:
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Inducible expression systems: Conditional expression systems using IPTG-inducible promoters (such as Ptac) have been successfully implemented in A. tumefaciens and could be adapted for controlled YidC expression.
What purification strategies yield the highest quality recombinant YidC protein?
Based on successful membrane protein purification approaches, consider:
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Membrane fraction isolation: Optimize protocols for A. tumefaciens's unique cell envelope structure, which bears similarities to its Rhizobium relatives .
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Detergent screening: While 1% DDM has proven effective for YidC extraction , a systematic detergent screen is recommended for A. tumefaciens YidC to maximize yield and activity.
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Affinity purification: Design constructs with appropriate affinity tags that don't interfere with YidC function, potentially positioning tags at the C-terminus as successfully demonstrated with BioID fusions .
