860 CYSTINOSIS: DEFECTIVE FIBROBLAST LYSOSOMAL CYSTINE TRANSPORT

Abstract

Defective cystine efflux has been demonstrated in lysosomes from both peripheral leukocytes and cultured EBV-lymphocytes from cystinotic patients. However, this abnormality has been difficult to demonstrate in fibroblast lysosomes, raising the question of whether lysosomal transport is the only defect in cystinosis. We have substantiated that some previous methods of loading fibroblast lysosomes with cystine, using cystine dimethyl ester (CDME), lead to cystine accumulation in a non-lysosomal as well as the lysosomal compartment (E. Harms, personal communication). If fibroblasts are removed from their anchorage substrate and incubated with 0.25 mM CDME for 15 min., only lysosomes are loaded with cystine as shown on Percoll gradients. In cystine-loaded lysosomes from two normal control fibroblast cultures the 20 min. cystine efflux was 19% and 20% without ATP and 56% and 74% with 2 mM MgATP. Lysosomes from two cystinotic fibroblast cultures had no cystine efflux over 20 min. both with and without ATP. We have used the dye, dipropylthiodicarbocyanine iodide to measure lysosomal membrane potential. MgATP causes a positive shift in membrane potential, in both cystinotic and normal lysosomes. In both cases, inhibitor and anion effects are consistent with an electrogenic proton pump. All of these findings support the concept of an abnormal lysosomal cystine transport protein as the primary defect in this disease.