Autolysosomal membrane-associated betaine homocysteine methyltransferase -Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy

We compared the membrane proteins of autolysosomes isolated from leupeptin- administered rat Liver with those of lysosomes. In addition to many polypep tides common to the two membranes, the autolysosomal membranes were found t o be more enriched in endoplasmic reticulum lumenal proteins (protein-disul fide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation -independent mannose g-phosphate receptor, transferrin receptor, Golgi 58-k Da protein) than lysosomal membranes. The autolysosomal membrane proteins i nclude three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequenc es have not yet been reported. Combining immunoblotting and reverse transcr iptase-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 au tolysosomes 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 pre sence of E64d, showing that the 32-kDa peptide is formed from the sequester ed 44-kDa peptide during autophagy. The accumulation is induced by rapamyci n but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a s treamlined assay for monitoring autophagy.