Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.
The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University.
Y
Y
Tanaka
R
R
Harada
M
M
Himeno
K
K
Kato
D000135:Acid Phosphatase / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002474:Cell-Free System; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006735:Horseradish Peroxidase; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009439:Neuraminidase; D017830:Octoxynol; D011061:Poly A / Q000378:metabolism; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D012156:Reticulocytes / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D011061:Poly A; D011092:Polyethylene Glycols; D012313:RNA; D017830:Octoxynol; C010615:Nonidet P-40; D006735:Horseradish Peroxidase; D000135:Acid Phosphatase; D006026:Glycoside Hydrolases; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase