Recombinant Dictyostelium discoideum UPF0041 protein A (DDB_G0267508)

What expression systems are optimal for DDB_G0267508 production?

Based on commercial production methodologies, E. coli has been successfully used as an expression host for recombinant DDB_G0267508 . The protein can be expressed as a full-length construct (1-97 amino acids) with an N-terminal His-tag. For optimal expression:

• Use BL21(DE3) or equivalent E. coli strains

• Clone the coding sequence into a pET vector system

• Induce expression with IPTG under controlled temperature conditions

• Purify using immobilized metal affinity chromatography (IMAC)

For membrane proteins like DDB_G0267508, inclusion of detergents during purification may be necessary to maintain proper folding and solubility.

What are the recommended storage conditions for recombinant DDB_G0267508?

For optimal stability and activity of recombinant DDB_G0267508:

• Store lyophilized powder at -20°C/-80°C upon receipt

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot and store at -20°C/-80°C for long-term storage

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

The protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during freeze-thaw cycles .

How can researchers validate the functional activity of recombinant DDB_G0267508?

As a probable mitochondrial pyruvate carrier, functional validation should focus on transport activity:

• Reconstitution assays: Incorporate purified DDB_G0267508 into liposomes and measure pyruvate transport using radioactive tracers or fluorescent pyruvate analogs

• Complementation studies: Express DDB_G0267508 in yeast MPC1-deficient strains and assess restoration of growth on pyruvate-dependent media

• Mitochondrial import assays: Confirm localization to mitochondria using isolated mitochondria and in vitro import assays

• Metabolic flux analysis: Measure changes in pyruvate metabolism using stable isotope tracers when DDB_G0267508 is present versus absent

• Binding studies: Assess interaction with known MPC complex components using co-immunoprecipitation or surface plasmon resonance

These approaches provide complementary evidence for the predicted pyruvate carrier function.

What genetic manipulation strategies are effective for studying DDB_G0267508 in vivo?

Dictyostelium discoideum is a well-established model organism with developed genetic tools . For studying DDB_G0267508:

• Gene knockout approaches:

  • Homologous recombination with selection markers

  • CRISPR/Cas9-mediated gene editing

  • Restriction enzyme-mediated integration (REMI)

• Expression modulation:

  • Antisense RNA or RNAi for knockdown

  • Inducible promoter systems for controlled expression

  • Fusion with fluorescent proteins for localization studies

• Phenotypic analysis:

  • Growth rate measurements under different carbon sources

  • Developmental timing during starvation-induced aggregation

  • Mitochondrial function assays (oxygen consumption, membrane potential)

The social amoeba's unique life cycle allows for studying the protein's role during both unicellular and multicellular developmental stages .

How does DDB_G0267508 relate to Dictyostelium's unique proteome characteristics?

Dictyostelium discoideum possesses "the highest content of prion-like proteins of all organisms investigated to date" . This unique proteome characteristic raises important questions about DDB_G0267508:

• Aggregation propensity: Though not specifically identified as prion-like in the search results, DDB_G0267508 exists in an organism that has evolved specialized mechanisms to manage highly aggregation-prone proteins

• Proteostasis interactions: DDB_G0267508 may interact with the robust chaperone systems that Dictyostelium employs to maintain proteome solubility

• Functional regulation: The protein may be subject to unique post-translational modifications or regulatory mechanisms specific to Dictyostelium's proteostasis network

Research approaches to investigate these aspects include:

• Computational prediction of aggregation propensity

• Co-immunoprecipitation with known proteostasis factors

• Stress response studies under conditions that compromise proteostasis

How does DDB_G0267508 compare with UPF0041 protein B (DDB_G0268478)?

Dictyostelium discoideum contains both UPF0041 protein A (DDB_G0267508) and UPF0041 protein B (DDB_G0268478). Key comparative features include:

These two proteins likely represent paralogs with potentially overlapping but distinct functions in Dictyostelium metabolism or development. Comparative functional studies could reveal whether they have redundant roles or specialized functions.

What is the evolutionary significance of DDB_G0267508 in relation to the Dictyostelium kinome?

While DDB_G0267508 itself is not identified as a kinase in the search results, understanding its evolutionary context within Dictyostelium is important:

• Dictyostelium discoideum diverged after the plant/animal split but before the divergence of fungi

• The Dictyostelium kinome shows both conserved and unique features compared to other organisms, reflecting its evolutionary position

• Analysis of the DDB_G0267508 sequence and function in comparison with homologs from animals, plants, and fungi could provide insights into the evolution of mitochondrial pyruvate transport

• The protein may represent an ancient form of pyruvate carrier that predates the divergence of the major eukaryotic lineages

Comparative genomic analysis across diverse taxa would help establish the evolutionary history and conservation of this important metabolic component.

How does DDB_G0267508 function integrate with Dictyostelium metabolic networks?

As a probable mitochondrial pyruvate carrier, DDB_G0267508 would occupy a critical position in cellular metabolism:

• Central carbon metabolism:

  • Controls pyruvate entry into mitochondria for TCA cycle

  • Influences the balance between respiration and fermentation

  • Acts as a regulatory point for carbon flux distribution

• Developmental metabolism:

  • May play a role in metabolic reprogramming during Dictyostelium's transition from unicellular to multicellular stages

  • Could influence energy availability during different developmental phases

• Stress response:

  • Potentially involved in metabolic adaptation to environmental challenges

  • May participate in coordinating respiratory capacity with nutrient availability

Systems biology approaches such as metabolic flux analysis, transcriptome profiling across developmental stages, and protein interaction mapping would help elucidate the broader functional context of DDB_G0267508.

What experimental approaches can resolve contradictions in DDB_G0267508 functional annotations?

The protein is annotated as both "UPF0041 protein A" (suggesting unknown function) and "probable mitochondrial pyruvate carrier 1" (suggesting a specific transport function) . To resolve this contradiction:

• Transport assays:

  • Liposome reconstitution with purified protein

  • Measurement of pyruvate transport using isotope-labeled substrates

  • Competition studies with known MPC inhibitors

• Structural analysis:

  • Homology modeling based on known MPC structures

  • Identification of conserved residues important for transport

  • Mutagenesis of predicted functional sites

• Transcriptional co-regulation:

  • Analysis of expression patterns with known metabolic genes

  • Identification of regulatory elements in the promoter region

  • Response to metabolic perturbations

• Phenotypic characterization:

  • Growth on different carbon sources

  • Mitochondrial function in knockout strains

  • Metabolomic profiling

This multifaceted approach would provide convergent evidence for the true biological function of DDB_G0267508.

How can DDB_G0267508 contribute to understanding Dictyostelium as a model organism?

Dictyostelium discoideum is a well-established model organism for studying various biological processes . Research on DDB_G0267508 could advance our understanding in several areas:

• Mitochondrial biology: As a potential mitochondrial carrier, DDB_G0267508 could provide insights into the evolution of energy metabolism in eukaryotes

• Developmental regulation: Studying its expression and function during Dictyostelium's life cycle could reveal connections between metabolism and development

• Proteostasis mechanisms: Given Dictyostelium's unique prion-like proteome , examining how DDB_G0267508 is maintained in a functional state could illuminate general principles of protein quality control

• Host-pathogen interactions: Dictyostelium is used as a model to study phagocyte-pathogen interactions , and metabolic regulators like DDB_G0267508 may influence these processes

What technological advances would enhance research on DDB_G0267508?

To advance our understanding of DDB_G0267508, several emerging technologies and approaches would be valuable:

• Cryo-electron microscopy: For determining the structure of DDB_G0267508 and its potential complexes at near-atomic resolution

• Single-cell omics: To analyze the heterogeneity of DDB_G0267508 expression and function during development and under different conditions

• Genome-wide interaction screens: To identify genetic interactions that modify DDB_G0267508 function or compensate for its loss

• In situ structural biology: Techniques like cryo-electron tomography could visualize DDB_G0267508 in its native mitochondrial environment

• Microfluidic phenotyping: For high-throughput analysis of mutant phenotypes under precisely controlled conditions

These advanced approaches would complement traditional biochemical and genetic methods to provide a comprehensive understanding of this protein's biological roles.