Biosythesis processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes

Yoshitaka Tanaka, Ryoko Harada, Masaru Himeno, Keitaro Kato

研究成果: ジャーナルへの寄稿記事

22 引用 (Scopus)

抄録

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-β-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 chioro quine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphata.se to lysosomes. Acid phosphat did not contain the phosphorylated high mannose type of ollgosaccharide 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 cia and trans Golgi elements, respectively, at 30 mm 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. At 3 h after the injection, acid phos phatases of 64 and 55 kDa were detected In lysosomal content fractions. Moreover, at 12 h after the injection, an acid phosphatase of 48 kDa, as well as the 55 kDa form, was present in the lysosomal content fraction, thereby indicating that after transport to the lysosomes, the membrane-bound acid phosphatase of 67 kDa was released into the lysosomal matrix, as the 64 kDa form, and was subsequently processed to the 55 kDa form and then converted into the 48 kDa form. The processing of the 67 kDa membrane-bound form to the 48 kDa soluble form involved removal of the protein portion and of the carbohydrate moieties. These results are Interpreted to mean that acid phosphatase is targeted to the lysosomes in a manner independent of the mannose-6-phosphate system.

元の言語英語
ページ(範囲)278-286
ページ数9
ジャーナルJournal of biochemistry
108
発行部数2
DOI
出版物ステータス出版済み - 1 1 1990

Fingerprint

Acid Phosphatase
Rats
Hepatocytes
Lysosomes
Processing
Membranes
Cell-Free System
Fractionation
Methionine
Labeling
Acids
trans-Golgi Network
Tunicamycin
Acetylglucosaminidase
Cathepsin D
Peptides
Injections
Experiments
Biosynthesis
Hydrolases

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

これを引用

Biosythesis processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes. / Tanaka, Yoshitaka; Harada, Ryoko; Himeno, Masaru; Kato, Keitaro.

:: Journal of biochemistry, 巻 108, 番号 2, 01.01.1990, p. 278-286.

研究成果: ジャーナルへの寄稿記事

Tanaka, Yoshitaka ; Harada, Ryoko ; Himeno, Masaru ; Kato, Keitaro. / Biosythesis processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes. :: Journal of biochemistry. 1990 ; 巻 108, 番号 2. pp. 278-286.
@article{f6009d69090b4bb3869cabd40517a9f7,
title = "Biosythesis processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes",
abstract = "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-β-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 chioro quine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphata.se to lysosomes. Acid phosphat did not contain the phosphorylated high mannose type of ollgosaccharide 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 cia and trans Golgi elements, respectively, at 30 mm 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. At 3 h after the injection, acid phos phatases of 64 and 55 kDa were detected In lysosomal content fractions. Moreover, at 12 h after the injection, an acid phosphatase of 48 kDa, as well as the 55 kDa form, was present in the lysosomal content fraction, thereby indicating that after transport to the lysosomes, the membrane-bound acid phosphatase of 67 kDa was released into the lysosomal matrix, as the 64 kDa form, and was subsequently processed to the 55 kDa form and then converted into the 48 kDa form. The processing of the 67 kDa membrane-bound form to the 48 kDa soluble form involved removal of the protein portion and of the carbohydrate moieties. These results are Interpreted to mean that acid phosphatase is targeted to the lysosomes in a manner independent of the mannose-6-phosphate system.",
author = "Yoshitaka Tanaka and Ryoko Harada and Masaru Himeno and Keitaro Kato",
year = "1990",
month = "1",
day = "1",
doi = "10.1093/oxfordjournals.jbchem.a123194",
language = "English",
volume = "108",
pages = "278--286",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "2",

}

TY - JOUR

T1 - Biosythesis processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes

AU - Tanaka, Yoshitaka

AU - Harada, Ryoko

AU - Himeno, Masaru

AU - Kato, Keitaro

PY - 1990/1/1

Y1 - 1990/1/1

N2 - 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-β-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 chioro quine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphata.se to lysosomes. Acid phosphat did not contain the phosphorylated high mannose type of ollgosaccharide 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 cia and trans Golgi elements, respectively, at 30 mm 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. At 3 h after the injection, acid phos phatases of 64 and 55 kDa were detected In lysosomal content fractions. Moreover, at 12 h after the injection, an acid phosphatase of 48 kDa, as well as the 55 kDa form, was present in the lysosomal content fraction, thereby indicating that after transport to the lysosomes, the membrane-bound acid phosphatase of 67 kDa was released into the lysosomal matrix, as the 64 kDa form, and was subsequently processed to the 55 kDa form and then converted into the 48 kDa form. The processing of the 67 kDa membrane-bound form to the 48 kDa soluble form involved removal of the protein portion and of the carbohydrate moieties. These results are Interpreted to mean that acid phosphatase is targeted to the lysosomes in a manner independent of the mannose-6-phosphate system.

AB - 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-β-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 chioro quine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphata.se to lysosomes. Acid phosphat did not contain the phosphorylated high mannose type of ollgosaccharide 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 cia and trans Golgi elements, respectively, at 30 mm 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. At 3 h after the injection, acid phos phatases of 64 and 55 kDa were detected In lysosomal content fractions. Moreover, at 12 h after the injection, an acid phosphatase of 48 kDa, as well as the 55 kDa form, was present in the lysosomal content fraction, thereby indicating that after transport to the lysosomes, the membrane-bound acid phosphatase of 67 kDa was released into the lysosomal matrix, as the 64 kDa form, and was subsequently processed to the 55 kDa form and then converted into the 48 kDa form. The processing of the 67 kDa membrane-bound form to the 48 kDa soluble form involved removal of the protein portion and of the carbohydrate moieties. These results are Interpreted to mean that acid phosphatase is targeted to the lysosomes in a manner independent of the mannose-6-phosphate system.

UR - http://www.scopus.com/inward/record.url?scp=0025266607&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025266607&partnerID=8YFLogxK

U2 - 10.1093/oxfordjournals.jbchem.a123194

DO - 10.1093/oxfordjournals.jbchem.a123194

M3 - Article

C2 - 1699935

AN - SCOPUS:0025266607

VL - 108

SP - 278

EP - 286

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 2

ER -