Abstract
Purpose: :
Folate is essential for de novo synthesis of DNA and certain amino acids. It is a lipophobic, bivalent anion; hence, all tissues require transport mechanisms to acquire folate. Previously, we localized folate receptor α (FRα) and reduced-folate transporter-1 (RFT-1) to the basolateral and apical RPE surfaces, respectively (Chancy et al, JBC, 2000). FRα and RFT-1 work coordinately to facilitate folate travel across the outer blood-retinal barrier. Additionally, FRα is expressed ubiquitously in the neural retina (ganglion cell layer (GCL), inner nuclear layer (INL), photoreceptor inner segments (IS)) (Smith et al, IOVS, 1999). Folate entry into cells via FRα occurs by receptor-mediated endocytosis; however, the mechanism by which folate is delivered from the endosome into the cytoplasm is not known. Recently, a new folate transporter, PCFT (Mr ~55kD), was identified in duodenum (Qiu et al, Cell, 2006). We hypothesized that PCFT is the transporter by which folate is released from the endosome. Initial studies to test this hypothesis analyzed PCFT mRNA expression in retina (Umapathy et al, IOVS, 2007). In the current study, we examined PCFT protein expression in retina.
Methods: :
An antibody against PCFT was generated and affinity-purified and gave results similar to those obtained using the antibody of Qui et al (2006). The purified antibody was used for (1) immunoblotting of mouse neural retina and RPE/eyecup, (2) immunohistochemistry (IHC) of intact mouse retina and (3) immunocytochemistry (ICC) of primary mouse ganglion (GC) and Müller cells (MC).
Results: :
Immunoblotting showed that PCFT is present in neural retina and RPE/eyecup. IMH showed that PCFT is present in the GCL, INL, IS, and RPE. ICC of GCs and MCs revealed PCFT expression on the plasma membrane and in the cytoplasm of these cells.
Conclusions: :
PCFT is present ubiquitously in the retina; its expression pattern closely mimics that of FRα. Its plasma membrane and cytoplasmic location are well suited to a role as an endosomal folate transporter.
Keywords: protein purification and characterization • nutritional factors