title: Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy.
abstract: We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.
mesh_terms: D000818:Animals; D001329:Autolysis / Q000201:enzymology*; D001483:Base Sequence; D050940:Betaine-Homocysteine S-Methyltransferase; D015180:Electrophoresis, Gel, Two-Dimensional; D011992:Endosomes / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D008099:Liver / Q000166:cytology; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar