In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase.
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December 2000
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Research Article|
December 08 2000
Autophagosome-associated variant isoforms of cytosolic enzymes
Monica FENGSRUD;
Monica FENGSRUD
*Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
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Camilla RAIBORG;
Camilla RAIBORG
*Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
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Trond Olav BERG;
Trond Olav BERG
*Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
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Per Eivind STRØMHAUG;
Per Eivind STRØMHAUG
*Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
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Takashi UENO;
Takashi UENO
†Department of Biochemistry, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
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Egil S. ERICHSEN;
Egil S. ERICHSEN
‡Laboratory for Electron Microscopy, Faculty of Science, University of Bergen, Allégaten 41, N-5007 Bergen, Norway
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Per O. SEGLEN
Per O. SEGLEN
1
*Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
1To whom correspondence should be addressed (e-mail per.seglen@labmed.uio.no).
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Publisher: Portland Press Ltd
Received:
April 28 2000
Revision Received:
August 21 2000
Accepted:
September 26 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 352 (3): 773–781.
Article history
Received:
April 28 2000
Revision Received:
August 21 2000
Accepted:
September 26 2000
Citation
Monica FENGSRUD, Camilla RAIBORG, Trond Olav BERG, Per Eivind STRØMHAUG, Takashi UENO, Egil S. ERICHSEN, Per O. SEGLEN; Autophagosome-associated variant isoforms of cytosolic enzymes. Biochem J 15 December 2000; 352 (3): 773–781. doi: https://doi.org/10.1042/bj3520773
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