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Journal of Biochemistry Advance Access published online on March 3, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn030
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© 2008 The Japanese Biochemical Society

Human calpain 7/PalBH associates with a subset of ESCRT-III-related proteins in its N-terminal region and partly localizes to endocytic membrane compartments

Chiharu Yorikawa1,#, Emi Takaya1, Yohei Osako1, Ryohei Tanaka1, Yoshinori Terasawa1, Takao Hamakubo2, Yasuhiro Mochizuki2, Hiroko Iwanari2, Tatsuhiko Kodama2, Tatsuya Maeda3, Kiyotaka Hitomi1, Hideki Shibata1 and Masatoshi Maki1,*

1Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
2Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan.
3Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

*To whom correspondence should be addressed: Dr. Masatoshi Maki, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Tel: +81-52-789-4088, Fax: +81-52-789-5542, E-mail: mmaki{at}agr.nagoya-u.ac.jp

Received December 20, 2007; Accepted February 13, 2008


  Abstract

Calpain 7 (also known as PalBH) is a mammalian homologue of the Aspergillus atypical calpain PalB. Knowledge of the biochemical properties of calpain 7 is limited and its function is not yet known. In this study, we investigated the interactions of calpain 7 with all eleven ESCRT-III-related proteins, named charged multivesicular body proteins (CHMPs), and the subcellular localization of calpain 7. Pulldown assays using stable HEK293T transfectants of Strep-tagged calpain 7 revealed interactions of calpain 7 with a subset of FLAG-tagged CHMPs, among which CHMP1B was selected for further analyses. The N-terminal region containing a tandem repeat of MIT domains of calpain 7 was found to be necessary and sufficient for interaction with CHMP1B. Direct interaction was confirmed by a pulldown assay using recombinant proteins. Fluorescence microscopic analysis using HeLa cells revealed that overexpression of GFP-fused CHMPs or a dominant-negative construct of SKD1/Vps4B caused accumulation of epitope-tagged calpain 7 in a punctate pattern in the perinuclear area. Subcellular fractionation revealed that the most of endogenous calpain 7 is present in the cytosol but a small portion is present in particulate fractions. Punctate fluorescence signals of monomeric GFP-fused calpain 7 partly merged with those of endocytosed tetramethylrhodamine-labeled EGF. These results suggest that calpain 7 plays roles in the endosomal pathway by interacting with a subset of ESCRT-III-related proteins.

Key Words: ESCRT, calpain, CHMP, endosome, MIT domain

# JSPS research fellow


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