Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function.
The reduced folate carrier (RFC) protein has a secondary structure consistent with the predicted 12 transmembrane (TM) domains, intracellular N- and C-termini and a large cytoplasmic loop between TM6 and TM7. In the present study, the role of the cytoplasmic domains in substrate transport and protein biogenesis were examined using an array of hamster RFC deletion mutants fused to enhanced green fluorescent protein and expressed in Chinese hamster ovary cells. The N- and C-terminal tails were removed both individually and together, or the large cytoplasmic loop was modified such that the domain size and role of conserved sequences could be examined. The loss of the N- or C-terminal tails did not appear to significantly disrupt protein function, although both termini appeared to have a role in the efficiency with which molecules exited the endoplasmic reticulum to localize at the plasma membrane. There appeared to be both size and sequence requirements for the intracellular loop, which are able to drastically affect protein stability and function unless met. Furthermore, there might be an indirect role for the loop in substrate translocation, since even moderate changes significantly reduced the V(max) for methotrexate transport. Although these cytoplasmic domains do not appear to be absolutely essential for substrate transport, each one is important for biogenesis and localization.
Sadlish
Heather
H
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Williams
Frederick M R
FM
Flintoff
Wayne F
WF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019921:Biotinylation; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005492:Folic Acid