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J. Biol. Chem., Vol. 276, Issue 13, 9846-9854, March 30, 2001
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From the Apg7p/Cvt2p, a protein-activating enzyme, is
essential for both the Apg12p-Apg5p conjugation system and the Apg8p
membrane targeting in autophagy and cytoplasm-to-vacuole targeting in
the yeast Saccharomyces cerevisiae. Similar to the
ubiquitin-conjugating system, both Apg12p and Apg8p are activated by
Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an
E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred
to Apg3p, another E2-like enzyme, followed by conjugation with
phosphatidylethanolamine. Evidence is presented here that Apg7p
forms a homodimer with two active-site cysteine residues via the
C-terminal region. The dimerization of Apg7p is independent of the
other Apg proteins and facilitated by overexpressed Apg12p. The
C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is
neither an ATP binding domain nor an active-site cysteine essential for
its E1 activity. The deletion of its carboxyl 40 amino acids (residues
591-630 out of 630 amino acids) results in several defects of not only
Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for
Apg8p. These results suggest that Apg7p forms a homodimer via the
C-terminal region and that the C-terminal region is essential for both
the activity of the E1 enzyme for Apg12p and Apg8p as well as the
formation of an E1-E2 complex for Apg8p.
The C-terminal Region of an Apg7p/Cvt2p Is Required for
Homodimerization and Is Essential for Its E1 Activity and E1-E2
Complex Formation*
,,,
Department of Biochemistry, Juntendo
University School of Medicine, Tokyo 113-8421, § Department of Bioscience, Teikyo University of Science and
Technology, Yamanashi 409-0193, and ¶ Department of Cell
Biology, National Institute for Basic Biology,
Okazaki 444-8501, Japan
*
This work was supported in part by Grants-in-aid 12780543 (to I. T.), 09680629 (to T. U.), and 12470040 (to E. K.) for
Scientific Research, Grants-in-aid 12146205 (to E. K.) for Scientific
Research on Priority Areas from the Ministry of Education, Science,
Sports, and Culture of Japan, and The Science Research Promotion Fund from the Japan Private School Promotion Foundation (to E. K.).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 correspondence should be addressed. kominami@med.
juntendo.ac.jp.
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