Recombinant Schizosaccharomyces pombe Probable mitochondrial phosphate carrier protein (SPBC1703.13c)

What is SPBC1703.13c and what is its functional role in S. pombe?

SPBC1703.13c is classified as a probable mitochondrial phosphate carrier protein in fission yeast (Schizosaccharomyces pombe strain 972 / ATCC 24843). Based on homology to other mitochondrial carrier family proteins, it likely transports inorganic phosphate across the inner mitochondrial membrane. This function is similar to MIR1 and PIC2 in Saccharomyces cerevisiae, which are responsible for phosphate transport in baker's yeast . The protein's UniProt accession number is Q9P7V8 .

What commercial reagents are available for studying SPBC1703.13c?

Researchers can obtain a polyclonal antibody specifically targeting SPBC1703.13c (Product Code: CSB-PA889250XA01SXV). This antibody is:

• Raised in rabbit using recombinant SPBC1703.13c protein as the immunogen

• Suitable for ELISA and Western Blot applications

• Purified using antigen affinity methods

• Supplied in liquid form with 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as preservative

• Requires storage at -20°C or -80°C, avoiding repeated freeze-thaw cycles

• Available as a made-to-order product with 14-16 weeks lead time

How does SPBC1703.13c compare to phosphate transporters in other organisms?

The mitochondrial carrier family (MCF/SLC25) proteins responsible for phosphate transport show interesting evolutionary patterns. In mammals, SLC25A3 transports both copper and phosphate, while in S. cerevisiae these functions are partitioned between two paralogs: PIC2 (transports copper and possibly phosphate) and MIR1 (primary phosphate transporter) . Phylogenetic analyses indicate that PIC2-like and MIR1-like orthologs are present across all major eukaryotic supergroups, suggesting an ancient gene duplication event created these specialized transporters . SPBC1703.13c likely falls within this evolutionary framework, though its specific substrate preference (whether primarily phosphate or both phosphate and copper) would require experimental determination.

What structural features determine substrate specificity in SPBC1703.13c?

While specific structural data for SPBC1703.13c is not available in the search results, insights can be drawn from related transporters. MCF transporters generally consist of a conserved fold with three repeats containing two transmembrane helices connected by a short α-helical loop . Research on mouse SLC25A3 identified critical residues for substrate specificity—notably, an L175A variant retains copper transport ability while losing phosphate transport function .

To determine equivalent residues in SPBC1703.13c, researchers should:

• Perform sequence alignment with SLC25A3, PIC2, and MIR1

• Identify conserved residues in transmembrane domains

• Use site-directed mutagenesis to test the functional role of candidate residues

• Measure transport of both phosphate and potential alternative substrates like copper

Does SPBC1703.13c undergo alternative splicing that affects its function?

Alternative splicing is prevalent in S. pombe, with various types observed including intron retention, exon skipping, and novel exons . Though the search results don't specifically mention alternative splicing of SPBC1703.13c, researchers should investigate this possibility given examples of alternatively spliced genes in S. pombe with functional consequences.

For instance, SPBC1703.10 (ypt1), which shares the same chromosomal location prefix, exhibits exon inclusion alternative splicing events . To investigate potential alternative splicing in SPBC1703.13c, researchers could:

• Employ long-read sequencing (PacBio) to capture full-length transcripts

• Analyze mRNA expression patterns during different growth phases and stress conditions

• Use RT-PCR with primers designed to detect potential splice variants

• Examine temporal expression patterns, as some novel isoforms show distinct temporal regulation compared to annotated isoforms

How is SPBC1703.13c expression regulated at the transcriptional level?

The expression of phosphate-responsive genes in S. pombe (pho1, pho84, tgp1) is regulated by long noncoding RNAs (lncRNAs) transcribed from upstream flanking genes . This mechanism represents a unified model for the repressive arm of fission yeast phosphate homeostasis, where lncRNA transcription interferes with the promoters of the phosphate-responsive genes .

To determine if SPBC1703.13c is regulated similarly, researchers should:

• Identify potential lncRNAs in the genomic region upstream of SPBC1703.13c

• Analyze promoter elements (e.g., HomolD box, TATA box) that might be targeted by transcriptional interference

• Examine SPBC1703.13c expression under phosphate-replete versus phosphate-starved conditions

• Create reporter constructs with SPBC1703.13c promoter elements to identify regulatory regions

What phenotypes result from SPBC1703.13c deletion or mutation?

To characterize the functional importance of SPBC1703.13c, researchers should investigate the consequences of its deletion or mutation using the following approaches:

• Generate knockout strains using CRISPR/Cas9 or traditional homologous recombination

• Assess growth phenotypes under various conditions:

  • Different carbon sources (fermentable vs. non-fermentable)

  • Varying phosphate concentrations

  • Respiratory chain inhibitors

  • Copper availability (to test potential dual substrate specificity)

• Measure mitochondrial function parameters:

  • Oxygen consumption rate

  • Membrane potential

  • ATP production

• Perform complementation studies with orthologs (MIR1, PIC2, SLC25A3) to determine functional conservation

Can SPBC1703.13c transport substrates other than phosphate?

Based on the dual functionality of SLC25A3 (phosphate and copper transport) and the specialized roles of PIC2 (primarily copper) and MIR1 (primarily phosphate) in S. cerevisiae , researchers should investigate whether SPBC1703.13c can transport multiple substrates:

What are the optimal conditions for detecting SPBC1703.13c using Western blot?

When using the available polyclonal antibody (CSB-PA889250XA01SXV) :

• Sample preparation:

  • Use mitochondrial enrichment protocols to concentrate the target protein

  • Solubilize with appropriate detergents (e.g., digitonin, DDM) to maintain protein structure

  • Include protease inhibitor cocktail to prevent degradation

• Western blot conditions:

  • Protein load: 20-50 μg of mitochondrial protein per lane

  • Gel percentage: 10-12% SDS-PAGE for optimal resolution

  • Transfer: Semi-dry or wet transfer to PVDF membrane (preferred for hydrophobic proteins)

  • Blocking: 5% non-fat milk or BSA in TBST, 1 hour at room temperature

  • Primary antibody: Start with 1:1000 dilution, incubate overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP conjugate, 1:5000, 1 hour at room temperature

  • Detection: Enhanced chemiluminescence system

• Controls:

  • Positive control: Recombinant SPBC1703.13c protein

  • Negative control: Extract from SPBC1703.13c deletion strain

  • Loading control: Mitochondrial marker protein (e.g., porin)

How can recombinant SPBC1703.13c be expressed and purified for functional studies?

For successful expression and purification of this membrane protein:

• Expression system options:

  • E. coli with specialized strains (C41/C43) for membrane proteins

  • S. cerevisiae or P. pastoris for eukaryotic expression

  • Insect cell (baculovirus) system for higher yields of functional protein

• Vector design:

  • Include affinity tags (His6, FLAG) for purification

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Include a TEV protease site for tag removal

  • Use inducible promoters for controlled expression

• Purification strategy:

  • Gentle membrane solubilization with appropriate detergents

  • Affinity chromatography as initial capture step

  • Size exclusion chromatography for final polishing

  • Quality control by SDS-PAGE and activity assays

What techniques can determine if SPBC1703.13c is regulated by phosphate availability?

To investigate phosphate-dependent regulation:

• Transcriptional analysis:

  • qRT-PCR to measure SPBC1703.13c mRNA levels under varying phosphate conditions

  • RNA-seq to identify co-regulated genes and potential regulatory lncRNAs

  • 5' RACE to map transcription start sites under different conditions

• Promoter studies:

  • Reporter gene fusions (e.g., SPBC1703.13c promoter driving GFP expression)

  • Chromatin immunoprecipitation to identify transcription factor binding

  • Mutational analysis of predicted regulatory elements

• Protein expression analysis:

  • Western blotting with the available antibody under varying phosphate conditions

  • Pulse-chase experiments to determine protein half-life

  • Subcellular localization studies using tagged variants

How can transport activity of SPBC1703.13c be measured experimentally?

For functional characterization of transport activity:

• In vitro reconstitution system:

  • Purify SPBC1703.13c and reconstitute into proteoliposomes

  • Establish pH or ion gradients across the membrane

  • Measure uptake of radiolabeled phosphate (³²P)

  • Determine transport kinetics (Km, Vmax) and inhibitor sensitivity

• Whole-cell approaches:

  • Generate SPBC1703.13c overexpression and deletion strains

  • Measure ³²P uptake into intact cells or isolated mitochondria

  • Use phosphate-sensitive fluorescent probes to track subcellular phosphate distribution

  • Assess growth under phosphate-limited conditions

• Electrophysiological methods:

  • Patch-clamp of giant liposomes containing purified SPBC1703.13c

  • Planar lipid bilayer recordings to measure single-channel properties

  • Solid-supported membrane electrophysiology for pre-steady-state kinetics

What bioinformatic approaches can predict structural features of SPBC1703.13c?

In the absence of crystal structures, computational approaches can provide structural insights:

• Homology modeling:

  • Identify templates from related MCF proteins with known structures

  • Use tools like SWISS-MODEL, I-TASSER, or AlphaFold2

  • Validate models using energy minimization and structural evaluation tools

• Evolutionary analysis:

  • Multiple sequence alignment with functionally characterized orthologs

  • Identification of conserved residues across species

  • Correlation analysis to identify co-evolving residues that might be functionally linked

• Molecular dynamics:

  • Simulate protein behavior in a lipid bilayer environment

  • Identify potential substrate binding sites and transport pathways

  • Test effects of mutations observed in phylogenetic analysis