Cellular Distribution of Porphyrins In Porphyria Cutanea Tarda

Abstract 165

Porphyria cutanea tarda (PCT), the most common porphyria in humans, results from subnormal activity of uroporphyrinogen decarboxylase (URO-D) in hepatocytes. The enzymatic defects results in cellular accumulation of the highly carboxylated uroporphyrinogen and heptacarboxyl porphyrinogen. The excess porphyrinogens are oxidized to porphyrins, exported to plasma and eventually eliminated in the urine. The mechanism by which uroporphyrin and heptacarboxyl porphyrin are transported across cellular membranes is not known. We generated a mouse model of the familial form of PCT ¹. Mice heterozygous for a deletion of the Uro-d gene and homozygous for deletion of the hemochromatosis gene Hfe (genotype Uro-d^+/−;Hfe^−/−) accumulate hepatic porphyrins in a pattern similar to humans with PCT. Hepatocytes were isolated from porphyric mice using an in situ two-step EGTA/collagenase perfusion followed by an iso-density Percoll centrifugation step to remove damaged cells. Confocal microscopy was used to examine cells for porphyrin fluorescence by illuminating cells at 405 nm and monitoring the emission between 600 and 650 nm. Fluorescent porphyrins were present as punctate bodies within the cytosol of the hepatocytes. Co-staining with fluorescent probes (mitotracker and lysotracker) demonstrated co-localization of porphyrins with lysotracker, indicating that porphyrins were present in lysosomes. We utilized a yeast model to determine the molecular mechanism by which porphyrins are transferred from the cytosol to the lysosome. In S. cerevisiae the vacuole is analogous to the mammalian lysosome. The yeast homolog of Uro-d is HEM12. In a hem12 deletion strain, uroporphyrin accumulates in the vacuole. ABC transporters have previously been shown to transport tetrapyrroles across membranes. We used a candidate approach to identify the ABC transporter responsible for vacuolar transport of uroporphyrin by creating deletion strains of the five known vacuolar ABC transporters BPT1, YBT1, YCF1, VMR1 and NFT1. Vacuolar membranes were prepared from these strains by spheroplasting yeast with oxalyticase, osmotically lysing the cells and floating the vacuoles on a Ficoll gradient. Isolated vacuoles were assayed for tetrapyrrole uptake by incubating vacuoles (200 ug total protein), substrate (20 uM) and Mg²⁺-ATP (50 uM) at 30 °C for 1, 2, 4 and 8 min in 100 uL of import buffer (300 mM sorbitol, pH 6.8). As controls, reactions were carried out at 4 °C and in the absence of ATP. Vacuolar import of uroporphyrin was temperature-dependent (no import occurred at 4 °C) and was dependent on the presence of ATP. Uroporphyrinogen was imported but, the rate was approximately 10% that of uroporphyrin. No import of protoporphyrin IX was observed. Import of uroporphyrin into vacuoles from each of the ABC transporter mutants was essentially the same as wild-type vacuoles with the exception of the ycf1 deletion strain. There was no import of uroporphyrin or uroporphyrinogen in vacuoles from the ycf1 deletion stain. The mammalian ABC-type transporter with the highest homology to yeast YCF1 is ABCC1. ABCC1 has recently been shown to transport a tetrapyrrole (cobalamin) ² making ABCC1 a candidate transporter responsible for moving uroporphyrin across cellular membranes in humans.

1. Phillips JD, Jackson LK, Bunting M, et al. A mouse model of familial porphyria cutanea tarda. Proc Natl Acad Sci U S A. 2001;98:259-264.

2. Beedholm-Ebsen R, van de Wetering K, Hardlei T, Nexo E, Borst P, Moestrup SK. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin. Blood. 2010;115:1632-1639.