Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells.
YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.
Ren
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XQ
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima, 890-8520, Japan.
Furukawa
T
T
Chen
Z S
ZS
Okumura
H
H
Aoki
S
S
Sumizawa
T
T
Tani
A
A
Komatsu
M
M
Mei
X D
XD
Akiyama
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics*; D017997:Leukotriene C4 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C089179:YCF1 protein, S cerevisiae; D017997:Leukotriene C4; D000255:Adenosine Triphosphate