CLATHRIN HEAVY-CHAIN FUNCTIONS IN SORTING AND SECRETION OF LYSOSOMAL-ENZYMES IN DICTYOSTELIUM-DISCOIDEUM

The clathrin heavy chain is a major component of clathrin-coated vesic les that function in selective membrane traffic in eukaryotic cells. W e disrupted the clathrin heavy chain gene. (chcA) in Dictyostelium dis coideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a fo ur- to five-fold deficiency in the internalization of fluid-phase mark ers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of c lathrin heavy chain in the trafficking of lysosomal enzymes. Pulse cha se analysis revealed that clathrin-minus cells processed most alpha-ma nnosidase to mature forms, however, similar to 20-25 % of the precurso r molecules remained uncleaved, were missorted, and were rapidly secre ted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Stan dard secretion assays showed that the rate of secretion of alpha-manno sidase was significantly less in clathrin-minus cells compared to cont rol cells in growth medium. Interestingly, the secretion rates of anot her lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wildtype cells. Like wild-type cells, clathrin-minus mutants resp onded to starvation conditions with increased lysosomal enzyme secreti on. Our study of the mutant cells provide in vivo evidence for roles f or the clathrin heavy chain in (a) the internalization of fluid from t he plasma membrane; (b) sorting of hydrolase precursors from the const itutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.