Presence of detergent-resistant microdomains in lysosomal membranes.
We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.
Taute
Antje
A
Institut für Physiologische Chemie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 1, 35033, Marburg, Germany.
Wätzig
Kristin
K
Simons
Brigitte
B
Lohaus
Christiane
C
Meyer
Helmut
H
Hasilik
Andrej
A
D000123:Acetyltransferases / Q000032:analysis*; D002793:Cholic Acids / Q000737:chemistry; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D046911:Macromolecular Substances; D021962:Membrane Microdomains / Q000201:enzymology*; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D017830:Octoxynol / Q000737:chemistry
D002793:Cholic Acids; D003902:Detergents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D017830:Octoxynol; D000123:Acetyltransferases; C020580:glucosamine acetyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate