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.
Highlights
We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes
In a previous study [13], we found that isolated autolysosomal membranes possess two endoplasmic reticulum (ER) membrane proteins, cytochrome P450 and NADPH-cytochrome P450 reductase
As shown by two-dimensional gel electrophoresis, we were able to use lysosomal membranes isolated from dextran-loaded liver as a reference to identify autolysosomal polypeptides of nonlysosomal origin
Summary
We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In recent morphological studies on yeast autophagy [8, 9], autophagosomal membranes were found to have features distinct from those of other preexisting cell membranes. This appears to support the notion that the membrane may have a unique origin. In a previous study [13], we found that isolated autolysosomal membranes possess two ER membrane proteins, cytochrome P450 and NADPH-cytochrome P450 reductase These results are consistent with those of Dunn [5] in showing that autophagosomes originate from the ER. We systematically analyzed membrane proteins in isolated autolysosomes by two dimensional gel electrophoresis and compared the results with those of lysosomes isolated from dextran-loaded rat liver
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