Frank Tietze

The activity of a cystine transport system in lysosomes prepared from the leukocytes of patients with cystinosis was found to be deficient. In normal subjects, this system was resistant to N-ethylmaleimide and demonstrated saturation kinetics. Lysosomes from individuals heterozygous for cystinosis demonstrated a reduced maximum velocity for cystine egress from lysosomes. The rate of cystine escape from normal lysosomes was enhanced by adenosine triphosphate. The availability of normal and mutant lysosomes provides a means of investigating mechanisms of amino acid transport across lysosomal membranes.

Exposure of intact leucocytes to 0.25 m~ ~-[~~S]cystine dimethyl ester resulted in intralysosomal ester hydrolysis and free cystine accumulation in the isolated lysosome-rich granular fractions of normal, heterozygous, and cystinotic cells. Loaded normal and cystinotic granular fractions were incubated in 0.25 m~ sucrose, 10 m~ 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.0, at 37 “C under conditions in which lysosomal integrity, assessed by latency ofhexosaminidase, was substantially preserved. Serial aliquots were washed and treated with 10 m~ N-ethylmaleimide, and [35S]cystine and [3SS]cysteine-N-ethyImaleimide were analyzed by high voltage electrophoresis. Half-times for [36S]cystine loss from loaded cystinotic granular fractions (80.8 min f 10.7 S.E., N = 12) were much slower than normal (26.1 f 1.4, N = 13), with heterozygous intermediate (43.5 f 3.1, N = 8) (all p < 0.01). In contrast, for [35S]cysteine disposal, mean cystinotic TIf2 was 18.3 min f 0.9, heterozygous 16.9 zk 0.5, and normal 14.1 * 0.7. The rate of disposal of t3H] tryptophan from cystinotic granular fractions loaded with ~-[~H]tryptophan methyl ester (mean TlIz = 28.9 min) did not differ from normal (mean TlIz = 25.6 min). Similarly, ~-[~I€Jmethionine exodus from cystinotic granular fractions (mean = 17.5 min) was indistinguishable from normal (mean Tr,z = 17.2 min). Loading with unlabeled cystine dimethyl ester and serial assay of granular fraction cystine verified that cystinotic granular fractions fail to dispose of cystine. Under appropriate conditions, loss of cystine from normal granular fractions was quantitatively accounted for by cystine recovered in the medium. We conclude that isolated cystinotic lysosomes demonstrate a pronounced, selective defect in the exodus of cystine, but not of the other amino acids examined.

Normal fibroblasts exposed to N-acetylmannosamine yielded lysosome-rich granular fractions loaded with free (unbound) sialic acid, whose velocity of egress increased with increasing initial loading. Fibroblast granular fractions of patients with Salla disease exhibited negligible egress of sialic acid, whether endogenous or derived from N-acetylmannosamine exposure. Salla disease represents the first disorder demonstrated to be caused by defective transport of a monosaccharide out of cellular lysosomes.