Recombinant Schizosaccharomyces pombe Uncharacterized acyltransferase C1851.02 (SPAC1851.02)

Protein Overview

SPAC1851.02 (UniProt ID: Q9US20) is an uncharacterized acyltransferase in Schizosaccharomyces pombe. It functions as a 1-acyl-sn-glycerol-3-phosphate acyltransferase beta involved in sphingolipid metabolism. The protein consists of 279 amino acids with a molecular weight of 31.3 kDa and an isoelectric point of 9.9. It contains 2 transmembrane domains and is localized to the endoplasmic reticulum .

What is SPAC1851.02 and what is its basic function?

SPAC1851.02 (UniProt ID: Q9US20) is an uncharacterized acyltransferase in Schizosaccharomyces pombe. It functions as a 1-acyl-sn-glycerol-3-phosphate acyltransferase beta, which is primarily involved in sphingolipid metabolism and glycerophospholipid synthesis .

Methodology for functional characterization:

• Bioinformatic analysis using sequence similarity to known acyltransferases

• Domain identification using tools like Pfam and InterPro

• Basic biochemical assays to verify acyltransferase activity:

  • In vitro enzyme activity assays with labeled substrates

  • Thin-layer chromatography (TLC) to analyze reaction products

  • Western blotting for protein detection

What are the structural characteristics of SPAC1851.02?

SPAC1851.02 has the following structural characteristics:

Methodology for structural characterization:

• Primary structure determination:

  • 279 amino acids

  • Molecular weight: 31.3 kDa

  • Isoelectric point: 9.9

• Secondary and tertiary structure prediction:

  • Contains 2 transmembrane domains

  • Likely contains the HX4D catalytic motif common in acyltransferases

  • Membrane topology analysis suggests endoplasmic reticulum localization

• Experimental verification techniques:

  • Fluorescence microscopy with GFP-tagged protein

  • Differential interference contrast imaging

  • Proteomic analysis using mass spectrometry

How is SPAC1851.02 typically expressed and localized in S. pombe cells?

SPAC1851.02 is primarily localized to the endoplasmic reticulum membrane in S. pombe cells . The protein forms aggregates when expressed as a YFP fusion protein, suggesting possible oligomerization or interaction with other membrane components.

Methodology for expression and localization studies:

• Cultivation protocols:

  • Growth in YES (Yeast Extract with Supplements) or EMM (Edinburgh Minimal Medium)

  • Standard growth conditions: 30°C with shaking at 200 rpm

• Localization determination:

  • Fluorescent protein tagging (GFP or YFP fusion proteins)

  • Subcellular fractionation followed by Western blot analysis

  • Immunofluorescence microscopy with specific antibodies

• Expression analysis:

  • Quantitative PCR for mRNA levels

  • Western blotting for protein levels under different conditions

  • Promoter-reporter assays to study expression regulation

How does substrate specificity determine the function of SPAC1851.02 in lipid metabolism?

Substrate specificity is crucial for SPAC1851.02's function in lipid metabolism, particularly in determining which lipid species it produces. As a member of the acyltransferase family, it likely exhibits preferences for specific acyl-CoA donors and lysophospholipid acceptors that influence its role in sphingolipid metabolism.

Methodology for substrate specificity analysis:

• In vitro enzyme assays with multiple potential substrates:

  • Various acyl-CoA donors (differing in chain length and saturation)

  • Different lysophospholipid acceptors

  • Analysis of reaction products by LC-MS/MS

• Structure-function relationship studies:

  • Site-directed mutagenesis of putative substrate-binding residues

  • Enzyme kinetics to determine Km and Vmax for different substrates

  • Computational docking simulations to predict substrate binding

• Metabolic profiling in vivo:

  • Lipidomic analysis of wild-type vs. SPAC1851.02 deletion mutants

  • Stable isotope labeling to track metabolic flux

  • Complementation studies with mutated versions of the enzyme

Table 1: Predicted Substrate Preference of SPAC1851.02

What are the experimental approaches to characterize the enzymatic mechanism of SPAC1851.02?

Characterizing the enzymatic mechanism of SPAC1851.02 requires sophisticated experimental approaches that probe both structural and functional aspects. The conserved HX4D motif found in many acyltransferases is likely critical for its catalytic activity.

Methodology for enzymatic mechanism characterization:

• Active site identification:

  • Multiple sequence alignment with characterized acyltransferases

  • Conservation analysis to identify catalytic residues

  • Site-directed mutagenesis of predicted catalytic residues (especially the HX4D motif)

  • Activity assays with mutant proteins

• Reaction mechanism studies:

  • Pre-steady-state kinetics to identify rate-limiting steps

  • pH and temperature dependence studies

  • Isotope exchange experiments to determine reaction order

  • Inhibitor studies with transition state analogs

• Structural approaches:

  • X-ray crystallography or cryo-EM (challenging for membrane proteins)

  • Hydrogen-deuterium exchange mass spectrometry

  • Chemical crosslinking combined with mass spectrometry

  • NMR studies of substrate binding

Table 2: Predicted Activity of SPAC1851.02 Catalytic Mutants

ND: Not determinable due to very low activity

How do disruptions in SPAC1851.02 affect cellular sphingolipid homeostasis and membrane composition?

Disruptions in SPAC1851.02 can significantly impact cellular sphingolipid homeostasis and membrane composition, with downstream effects on cellular processes. By analogy with similar acyltransferases, deletion of SPAC1851.02 would likely alter the balance of lipid species in cellular membranes.

Methodology for studying disruption effects:

• Generation of deletion and conditional mutants:

  • CRISPR/Cas9 or homologous recombination for gene deletion

  • Tetracycline-inducible or temperature-sensitive mutants

  • Auxin-inducible degron system for controlled protein depletion

• Comprehensive lipidomic analysis:

  • Extraction of cellular lipids using Bligh and Dyer method

  • High-resolution mass spectrometry (UHPLC-MS/MS)

  • Quantitative analysis of sphingolipid species

  • Stable isotope labeling to track metabolic flux

• Membrane property assessments:

  • Fluorescence anisotropy to measure membrane fluidity

  • Atomic force microscopy to analyze membrane domains

  • Lipid raft isolation and characterization

  • Membrane protein activity assays

Table 3: Predicted Changes in Lipid Composition in SPAC1851.02Δ Mutant

What protein-protein interactions are critical for SPAC1851.02 function, and how can they be identified?

Protein-protein interactions are often crucial for enzyme function, regulation, and localization. For SPAC1851.02, identifying these interactions provides insight into its functional context within sphingolipid metabolism pathways.

Methodology for studying protein-protein interactions:

• Affinity purification-mass spectrometry (AP-MS):

  • Tandem affinity purification (TAP) tagging of SPAC1851.02

  • Gentle cell lysis to preserve protein complexes

  • Mass spectrometry identification of co-purified proteins

  • Quantitative comparison with control purifications

• Yeast two-hybrid assays:

  • Construction of bait (SPAC1851.02) and prey (S. pombe proteome) libraries

  • Screening for positive interactions by auxotrophic selection

  • Confirmation with secondary reporters (e.g., β-galactosidase)

  • Domain mapping to identify interaction regions

• In vivo validation:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Co-immunoprecipitation under native conditions

  • Functional studies of double mutants

Table 4: Potential SPAC1851.02 Protein Interaction Partners

Genetic Manipulation

• Transformation protocols using lithium acetate/PEG method for S. pombe

• Homologous recombination for gene deletion or modification

• CRISPR/Cas9 genome editing strategies

• Selection markers: ura4+, leu1+, kan, nat, hph

Protein Purification and Analysis

• Tandem affinity purification (TAP) protocol with calmodulin and IgG-binding tags

• Ni-NTA purification of His-tagged recombinant protein

• Blue native PAGE for membrane protein complexes

• Western blotting with specific antibodies

Lipid Analysis

• Extraction using Bligh and Dyer or Folch methods

• Thin-layer chromatography (TLC) for lipid class separation

• HPLC-MS/MS for detailed lipidomic analysis

• Radiolabeling with 14C-acetate or 3H-inositol to track lipid synthesis

Enzyme Activity Assays

• Spectrophotometric coupled enzyme assays

• Fluorescence-based activity assays with reporter substrates

• HPLC-based assays for direct product detection

• Radiolabeled substrate incorporation assays

Structural Biology Approaches

• Cryogenic electron microscopy (cryo-EM) to determine the 3D structure

• Molecular dynamics simulations to understand conformational changes

• Hydrogen-deuterium exchange mass spectrometry for dynamic structure analysis

Systems Biology Integration

• Integration of SPAC1851.02 function into lipid metabolism network models

• Multi-omics approaches (genomics, proteomics, lipidomics)

• Synthetic biology applications for engineered lipid production

Comparative Studies with Homologs

• Evolutionary analysis across yeast species

• Functional complementation with mammalian homologs

• Identification of conserved regulatory mechanisms