The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly345 → Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.
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Research Article|
February 22 2000
Mutations in the reduced-folate carrier affect protein localization and stability
Heather SADLISH;
Heather SADLISH
1Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
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Richard C. MURRAY;
Richard C. MURRAY
1Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
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Frederick M. R. WILLIAMS;
Frederick M. R. WILLIAMS
1Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
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Wayne F. FLINTOFF
Wayne F. FLINTOFF
1
1To whom correspondence and requests for offprints should be sent (e-mail flintoff@;julian.uwo.ca).
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Publisher: Portland Press Ltd
Received:
October 21 1999
Revision Received:
November 29 1999
Accepted:
December 22 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 346 (2): 509–518.
Article history
Received:
October 21 1999
Revision Received:
November 29 1999
Accepted:
December 22 1999
Citation
Heather SADLISH, Richard C. MURRAY, Frederick M. R. WILLIAMS, Wayne F. FLINTOFF; Mutations in the reduced-folate carrier affect protein localization and stability. Biochem J 1 March 2000; 346 (2): 509–518. doi: https://doi.org/10.1042/bj3460509
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