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Carrier-mediated transport of folate in a mutant of Pediococcus cerevisiae
Abstract:
A mutant strain of Pediococcus cerevisiae (P. cerevisiae/PteGlu) was isolated which grows on low-folate (PteGlu) concentrations (200 pg/ml). The growth response of the parent and mutant strains to folinate (5-CHO-H(4)PteGlu) was the same. The transport of (14)C-PteGlu by P. cerevisiae/PteGlu was temperature-dependent (Q(10) between 27 C and 37 C was about 2), energy-dependent, and pH-dependent and was inhibited by iodoacetate, 2,4-dinitrophenol, potassium fluoride, and sodium azide. The uptake obeyed saturation kinetics with an apparent K(m) of 6.6 x 10(-6) M and V(max) of 4.0 x 10(-10) mol per min per mg (dry weight). At the steady state the intracellular concentration of PteGlu was 120-fold higher from that of the medium. Reduced folates like 5-CHO-H(4)PteGlu and methyl-tetrahydrofolate (5-CH(3)-H(4)PteGlu) as well as 2,4-diaminoanalogues (amethopterin and aminopterin) were shown to compete for the PteGlue-carrier.
Insights
A mutant Pediococcus cerevisiae strain efficiently transports folate (PteGlu) via a temperature, energy, and pH-dependent carrier. This carrier exhibits saturation kinetics and concentrates folate intracellularly, showing competition with other folate analogs.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Folate (PteGlu) is essential for microbial growth and metabolic processes.
- Understanding folate transport mechanisms is crucial for microbial physiology and potential therapeutic interventions.
- A mutant strain of Pediococcus cerevisiae was developed to study folate uptake.
Purpose of the Study:
- To characterize the folate (PteGlu) transport system in a mutant Pediococcus cerevisiae strain.
- To investigate the kinetic and regulatory properties of the PteGlu carrier.
- To determine the substrate specificity of the folate transporter.
Main Methods:
- Isolation of a mutant Pediococcus cerevisiae strain (P. cerevisiae/PteGlu) exhibiting growth on low folate concentrations.
- Radiolabeled (14C-PteGlu) uptake studies to analyze transport kinetics.
- Assays for temperature, energy, and pH dependence, and inhibition studies using specific agents (iodoacetate, DNP, KF, NaN3).
- Competition assays with various folate analogs and antifolates.
Main Results:
- The mutant strain showed similar growth responses to folinate (5-CHO-H(4)PteGlu) as the parent strain.
- PteGlu transport was temperature-dependent (Q10 ≈ 2), energy-dependent, and pH-dependent.
- Uptake was inhibited by metabolic inhibitors, indicating an active transport process.
- The transporter exhibited saturation kinetics with an apparent Km of 6.6 x 10(-6) M and Vmax of 4.0 x 10(-10) mol/min/mg.
- Intracellular folate concentration reached 120-fold higher than extracellular levels.
- Reduced folates and 2,4-diaminoanalogues competed for the PteGlu carrier, suggesting broad substrate specificity.
Conclusions:
- Pediococcus cerevisiae possesses an active, carrier-mediated folate (PteGlu) transport system.
- The transporter is energy-dependent and exhibits Michaelis-Menten kinetics.
- The system concentrates folate intracellularly and shows affinity for various reduced folates and antifolates.