Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells.
BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.
Waheed
A
A
Zentrum Biochemie, Universität Göttingen, FRG.
Gottschalk
S
S
Hille
A
A
Krentler
C
C
Pohlmann
R
R
Braulke
T
T
Hauser
H
H
Geuze
H
H
von Figura
K
K
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008247:Lysosomes / Q000201:enzymology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D008565:Membrane Proteins; D011498:Protein Precursors; D004247:DNA; D000135:Acid Phosphatase