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Journal of Biochemistry Advance Access originally published online on March 3, 2008
Journal of Biochemistry 2008 143(6):731-745; 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; 2Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904; and 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. 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 11 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-labelled 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: calpain, CHMP, endosome, ESCRT, MIT domain

Abbreviations: CBB, Coomassie Brilliant Blue R-250; CHMP, charged multivesicular body protein; ESCRT, endosomal sorting complex required for transport; FBS, fetal bovine serum; GST, glutathione-S-transferase; MIT, microtubule interacting and trafficking; mAb, monoclonal antibody; mGFP, monomeric green fluorescent protein; mRFP, monomeric red fluorescent protein; MVB, multivesicular body; pAb, polyclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PEF, penta-EF-hand; Rh-EGF, tetramethylrhodamine-labelled epidermal growth factor; shRNA, short hairpin RNA; Trx, thioredoxin; Vps, vacuolar protein sorting; WB, western blotting


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