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 vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
T
T
Ruscetti
J A
JA
Cardelli
M L
ML
Niswonger
T J
TJ
O'Halloran
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002460:Cell Line; D002966:Clathrin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004271:DNA, Fungal / Q000032:analysis; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D005800:Genes, Fungal; D006867:Hydrolases / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000378:metabolism; D010873:Pinocytosis; D011498:Protein Precursors / Q000378:metabolism; D011995:Recombination, Genetic; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002966:Clathrin; D004271:DNA, Fungal; D011498:Protein Precursors; D006867:Hydrolases; D000135:Acid Phosphatase; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase