Lysosomal acid phosphatase is internalized via clathrin-coated pits.
The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.
Hille
A
A
Biochemie II, Universität Göttingen, Deutschland.
Klumperman
J
J
Geuze
H J
HJ
Peters
C
C
Brodsky
F M
FM
von Figura
K
K
D000135:Acid Phosphatase / Q000378:metabolism*; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002478:Cells, Cultured / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008845:Microinjections; D011506:Proteins / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D002966:Clathrin; D011506:Proteins; D000135:Acid Phosphatase