Autolysosomal Membrane-associated Betaine Homocysteine Methyltransferase

doi:10.1074/jbc.274.21.15222

Autolysosomal Membrane-associated Betaine Homocysteine Methyltransferase

LIMITED DEGRADATION FRAGMENT OF A SEQUESTERED CYTOSOLIC ENZYME MONITORING AUTOPHAGY*

From the ^‡Department of Biochemistry and ^§Central Laboratory for Medical Sciences, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421 and the ^¶Department of Applied Biochemistry, Faculty of Agriculture, Niigata University, Igarashi, Niigata 950-21, Japan

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.

Footnotes

↵* This work was supported by Grant-in-aid for Scientific Research 09680629 and Grant-in-aid for Scientific Research on Priority Areas (Intracellular Proteolysis) 08278103 from the Ministry of Education, Science, Sports and Culture of Japan and the Science Research Promotion Fund from the Japan Private School Promotion Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom all correspondence should be addressed: Dept. of Biochemistry, Juntendo University School of Medicine, Bldg. 9, Rm. 913, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: 813-5802-1031; Fax: 813-5802-5889; E-mail: kominami{at}med.juntendo.ac.jp.
Abbreviations:

ER

endoplasmic reticulum

FCS

fetal calf serum

PAGE

polyacrylamide gel electrophoresis

CI-M6PR

cation-independent mannose 6-phosphate receptor

E64c

(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylic acid

E64d

ethyl-(+)-(2S,3S)-3-[(S)-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylate

Caps

3-cyclo- hexylaminopropanesulfonic acid

Tes

N-tris(hydroxymethyl)-2aminoethanesulfonic acid

PVDF

polyvinylidene fluoride

BiP

immunoglobulin heavy chain binding protein

BHMT

betaine homocysteine methyltransferase

α-p44–10R

antibody raised against the NH₂terminal 10 amino acid residues (APIAGKKAKR) of the intact subunit of BHMT

α-p35–10R

antibody raised against the NH₂-terminal 10 amino acid residues (YVAEKISGQK) of the 35-kDa fragment of BHMT

α-p35–5R

antibody raised against the NH₂-terminal 5 amino acid residues (YVAEK) of the 35-kDa fragment of BHMT

α-p32–10R

antibody raised against the NH₂-terminal 10 amino acid residues (KISGQKVNEA) of the 32-kDa fragment of BHMT

α-p32–5R

antibody raised against the NH₂-terminal 5 amino acid residues (KISGQ) of the 32-kDa fragment of BHMT

KRB

Krebs-Ringer bicarbonate

RACE

rapid amplification of cDNA ends

RT

reverse transcriptase

PCR

polymerase chain reaction
- Received January 11, 1999.
- Revision received March 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.