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Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells
X Q Ren1, T Furukawa, Z S Chen
1Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima, 890-8520, Japan.
Biochemical and Biophysical Research Communications
|February 7, 2001
Summary
Yeast vacuole transporter YCF1 and multidrug resistance protein MRP1 share similarities. Expressing YCF1 in insect cells revealed functional parallels and differences with MRP1, aiding structure-function studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Yeast vacuole membrane transporter YCF1 confers resistance to cadmium and glutathione conjugates.
- Multidrug resistance protein 1 (MRP1) is implicated in multidrug resistance (MDR) in tumor cells.
- YCF1 and MRP1 exhibit significant amino acid sequence homology (63% similarity).
Purpose of the Study:
- To investigate the structure-function relationships of YCF1 and MRP1.
- To compare the functional characteristics of YCF1 and MRP1 when expressed in a heterologous system.
Main Methods:
- YCF1 and MRP1 were expressed with His epitopes in Sf21 insect cells.
- Membrane vesicles from Sf21 cells expressing YCF1 (Sf/YCF1-His) were used for transport assays.
- ATP-dependent transport of [3H]LTC(4) was measured, and kinetics were analyzed.
- Inhibition studies were performed using MDR reversing agents like MK-571, ONO-1078, PAK-104P, and KE1.
Main Results:
- Both YCF1 and MRP1 were localized to the plasma membrane in Sf21 insect cells, differing from YCF1's yeast localization.
- Sf/YCF1-His vesicles demonstrated ATP-dependent, osmotically sensitive, and saturable transport of LTC(4) with kinetics similar to yeast.
- YCF1 exhibited a higher K(m) for LTC(4) (758 nM) compared to MRP1 (108 nM).
- MK-571 and ONO-1078 inhibited LTC(4) transport by both YCF1 and MRP1.
- PAK-104P and KE1 showed differential inhibition effects on YCF1 and MRP1 transport activity.
Conclusions:
- Yeast YCF1 was successfully expressed in Sf21 insect cells, with altered cellular localization.
- YCF1 function in insect cells shares similarities but is not identical to MRP1 function.
- These findings provide insights into the structure-function relationships of YCF1 and MRP1.