Molecular and biochemical characterization of folate transport proteins in retinal müller cells

B. Renee Bozard, Preethi S. Ganapathy, Jennifer Duplantier, Barbara Mysona, Yonju Ha, Penny Roon, Robert Smith, I. David Goldman, Puttur Prasad, Pamela M. Martin, Vadivel Ganapathy, Sylvia B. Smith

Research output: Contribution to journalArticle

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Abstract

Purpose. To analyze the mechanisms of folate uptake in retinal Muller cells. Methods. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Muller cells, and rMC-1 cells for the three known folate transport proteins folate receptor α (FRα), 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 FRα and PCFT in primary Muller cells. The pH dependence of the uptake of [3H]-methyltetrahydrofolate ([3H]-MTF) was assayed in Muller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. Results. FRα and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Muller 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 FRα and PCFT on the plasma membrane and nuclear membrane and within en-dosomal structures. Muller cell uptake of [3H]-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 Muller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. Conclusions. FRα and PCFT are expressed in retinal Muller 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 Muller cells may reflect the upregulation of this protein under proliferative conditions.

Original languageEnglish (US)
Pages (from-to)3226-3235
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number6
DOIs
StatePublished - Jun 1 2010

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Folic Acid Transporters
Ependymoglial Cells
Proton-Coupled Folate Transporter
Reduced Folate Carrier Protein
Folic Acid
Nuclear Envelope
Confocal Microscopy
Retina
Cell Membrane
Thiamine Pyrophosphate
Proteins
Immunoelectron Microscopy
Cultured Cells
Up-Regulation

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Molecular and biochemical characterization of folate transport proteins in retinal müller cells. / Renee Bozard, B.; Ganapathy, Preethi S.; Duplantier, Jennifer; Mysona, Barbara; Ha, Yonju; Roon, Penny; Smith, Robert; David Goldman, I.; Prasad, Puttur; Martin, Pamela M.; Ganapathy, Vadivel; Smith, Sylvia B.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 6, 01.06.2010, p. 3226-3235.

Research output: Contribution to journalArticle

Renee Bozard, B. ; Ganapathy, Preethi S. ; Duplantier, Jennifer ; Mysona, Barbara ; Ha, Yonju ; Roon, Penny ; Smith, Robert ; David Goldman, I. ; Prasad, Puttur ; Martin, Pamela M. ; Ganapathy, Vadivel ; Smith, Sylvia B. / Molecular and biochemical characterization of folate transport proteins in retinal müller cells. In: Investigative Ophthalmology and Visual Science. 2010 ; Vol. 51, No. 6. pp. 3226-3235.
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abstract = "Purpose. To analyze the mechanisms of folate uptake in retinal Muller cells. Methods. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Muller cells, and rMC-1 cells for the three known folate transport proteins folate receptor α (FRα), 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 FRα and PCFT in primary Muller cells. The pH dependence of the uptake of [3H]-methyltetrahydrofolate ([3H]-MTF) was assayed in Muller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. Results. FRα and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Muller 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 FRα and PCFT on the plasma membrane and nuclear membrane and within en-dosomal structures. Muller cell uptake of [3H]-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 Muller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. Conclusions. FRα and PCFT are expressed in retinal Muller 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 Muller cells may reflect the upregulation of this protein under proliferative conditions.",
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T1 - Molecular and biochemical characterization of folate transport proteins in retinal müller cells

AU - Renee Bozard, B.

AU - Ganapathy, Preethi S.

AU - Duplantier, Jennifer

AU - Mysona, Barbara

AU - Ha, Yonju

AU - Roon, Penny

AU - Smith, Robert

AU - David Goldman, I.

AU - Prasad, Puttur

AU - Martin, Pamela M.

AU - Ganapathy, Vadivel

AU - Smith, Sylvia B.

PY - 2010/6/1

Y1 - 2010/6/1

N2 - Purpose. To analyze the mechanisms of folate uptake in retinal Muller cells. Methods. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Muller cells, and rMC-1 cells for the three known folate transport proteins folate receptor α (FRα), 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 FRα and PCFT in primary Muller cells. The pH dependence of the uptake of [3H]-methyltetrahydrofolate ([3H]-MTF) was assayed in Muller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. Results. FRα and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Muller 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 FRα and PCFT on the plasma membrane and nuclear membrane and within en-dosomal structures. Muller cell uptake of [3H]-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 Muller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. Conclusions. FRα and PCFT are expressed in retinal Muller 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 Muller cells may reflect the upregulation of this protein under proliferative conditions.

AB - Purpose. To analyze the mechanisms of folate uptake in retinal Muller cells. Methods. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Muller cells, and rMC-1 cells for the three known folate transport proteins folate receptor α (FRα), 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 FRα and PCFT in primary Muller cells. The pH dependence of the uptake of [3H]-methyltetrahydrofolate ([3H]-MTF) was assayed in Muller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. Results. FRα and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Muller 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 FRα and PCFT on the plasma membrane and nuclear membrane and within en-dosomal structures. Muller cell uptake of [3H]-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 Muller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. Conclusions. FRα and PCFT are expressed in retinal Muller 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 Muller cells may reflect the upregulation of this protein under proliferative conditions.

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