Autophagosome-associated variant isoforms of cytosolic enzymes.
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 31 kDa, 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.
Fengsrud
M
M
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Raiborg
C
C
Berg
T O
TO
Strømhaug
P E
PE
Ueno
T
T
Erichsen
E S
ES
Seglen
P O
PO
D000595:Amino Acid Sequence; D000818:Animals; D001124:Argininosuccinate Synthase / Q000737:chemistry / Q000378:metabolism; D001343:Autophagy; D048429:Cell Size; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D011992:Endosomes / Q000737:chemistry / Q000201:enzymology; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000378:metabolism; D005614:Freeze Fracturing; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010588:Phagosomes / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D007527:Isoenzymes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004746:Enoyl-CoA Hydratase; D001124:Argininosuccinate Synthase