ABNORMAL TRANSPORT ALONG THE LYSOSOMAL PATHWAY IN MUCOLIPIDOSIS, TYPE-IV DISEASE

Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disea se that is characterized by lysosomal accumulation of sphingolipids, p hospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we exa mined the hypothesis that the ML-IV phenotype might arise from abnorma l transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycli ng were nearly identical in ML-IV and normal fibroblasts. A fluorescen t analog of lactosylceramide (LacCer) was used to study plasma membran e lipid internalization and subsequent transport. Lipid internalizatio n at 19 degrees C was similar in both cell types; however, 40-60 min a fter raising the temperature to 37 degrees C, the fluorescent lipid ac cumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts, Biochemical studies demons trated that at these time points, hydrolysis of the lipid analog was m inimal (similar to 7%) in both cell types. A fluorescence ratio imagin g assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts, By 60 min, LacCer apparently decreased in the lys osomes of normal fibroblasts but not in ML-IV cells, suggesting that l ipid efflux from the lysosomes was also impaired. These results demons trate that there is a defect in ML-IV fibroblasts that affects membran e sorting and/or late steps of endocytosis.