Molecular and biochemical characterization of folate transport proteins in retinal Müller cells.
OBJECTIVE
To analyze the mechanisms of folate uptake in retinal Müller cells.


METHODS
RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Müller cells, and rMC-1 cells for the three known folate transport proteins folate receptor alpha (FRalpha), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Laser scanning confocal microscopy (LSCM) and immunoelectron microscopy were used to determine the subcellular location of FRalpha and PCFT in primary Müller cells. The pH dependence of the uptake of [(3)H]-methyltetrahydrofolate ([(3)H]-MTF) was assayed in Müller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC.


RESULTS
FRalpha and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Müller cells and rMC-1 cells. LSCM localized these proteins to the plasma membrane, nuclear membrane, and perinuclear region. Immunoelectron microscopic studies revealed the colocalization of FRalpha and PCFT on the plasma membrane and nuclear membrane and within endosomal structures. Müller cell uptake of [(3)H]-MTF was robust at pH 5.0 to 6.0, consistent with PCFT activity, but also at neutral pH, reflecting RFC function. RFC was expressed in mouse Müller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells.


CONCLUSIONS
FRalpha and PCFT are expressed in retinal Müller cells and colocalize in the endosomal compartment, suggesting that the two proteins may work coordinately to mediate folate uptake. The unexpected finding of RFC expression and activity in cultured Müller cells may reflect the upregulation of this protein under proliferative conditions.
Bozard
B Renee
BR
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Ganapathy
Preethi S
PS
Duplantier
Jennifer
J
Mysona
Barbara
B
Ha
Yonju
Y
Roon
Penny
P
Smith
Robert
R
Goldman
I David
ID
Prasad
Puttur
P
Martin
Pamela M
PM
Ganapathy
Vadivel
V
Smith
Sylvia B
SB
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism; D058975:Folate Receptor 1; D005786:Gene Expression Regulation / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D058979:Proton-Coupled Folate Transporter; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D058978:Reduced Folate Carrier Protein; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013763:Tetrahydrofolates / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000494:pharmacology
D058975:Folate Receptor 1; C520574:Folr1 protein, mouse; D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; C516877:Slc46a1 protein, mouse; D013763:Tetrahydrofolates; D013835:Thiamine Pyrophosphate; C005984:5-methyltetrahydrofolate