| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
The membrane proteins of autolysosomes isolated from leupeptin-administered rat liver have been extensively compared with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomaal membranes were found to bemore enriched in endoplasmic reticulum lumenal proteins (protein disulfide isomerase, caireticulin, ER6O, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58K) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44k, 35k, and 32k) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and RT-PCR analyses, we identified the 44k peptide as the intact subunit of betaine homocysteine methyltransferase and the 35k and 32k peptides as two proteolytic fragments. When freshly isolated autolysosomes were incubated with pronase at 0ﾟC, both p44 and p32 were resistant to the digestion whereas p35 was completely digested. It was concluded that the 44k and 32k peptides are present in the lumen, whereas the 35k peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32k peptide occurs in the presence of E64d, showing that the 32k peptide is formed from the sequestered 44k peptide during autophagy. The accumulation is enhanced by rapamycin but completely inhibited by wortmannin, 3- methyladenine, and bafilomycin. Thus, detection of the 32k peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.
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