Mutations in the reduced-folate carrier affect protein localization and stability

Area of Science:

• Cell Biology
• Genetics
• Molecular Biology

Background:

• The reduced-folate-carrier (rfc) gene is crucial for transporting reduced folate in mammalian cells.
• Methotrexate transport is essential for cellular folate metabolism and drug efficacy.

Purpose of the Study:

• To identify and characterize mutations in the rfc gene of a methotrexate-resistant Chinese hamster ovary (CHO) cell line.
• To understand the molecular mechanisms underlying the loss of reduced-folate transport in this mutant cell line.

Main Methods:

• Genetic analysis of the rfc gene in a mutant CHO cell line.
• Identification of point mutations and splice site alterations in both rfc alleles.
• Monitoring of altered gene products using green fluorescent protein (GFP) fusion.
• Analysis of protein localization, stability, and degradation pathways.

Main Results:

• Two distinct mutations were found in the rfc gene: a Gly(345)-->Arg substitution and a splice site mutation in intron 5.
• The Gly(345)-->Arg mutation results in a core-glycosylated, immature protein trapped in the endoplasmic reticulum (ER).
• The splice mutation leads to a truncated, non-membrane-associated protein with altered C-terminal amino acids.
• Both mutant rfc proteins are non-functional, exhibit increased turnover, and are degraded, explaining the loss of methotrexate transport.

Conclusions:

• Mutations in both alleles of the rfc gene are responsible for the observed deficiency in reduced-folate transport.
• The study provides insights into protein quality control mechanisms and degradation pathways within the ER.
• These findings underscore the critical role of the rfc gene in cellular folate homeostasis.