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.
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.
Yorikawa
Chiharu
C
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Takaya
Emi
E
Osako
Yohei
Y
Tanaka
Ryohei
R
Terasawa
Yoshinori
Y
Hamakubo
Takao
T
Mochizuki
Yasuhiro
Y
Iwanari
Hiroko
H
Kodama
Tatsuhiko
T
Maeda
Tatsuya
T
Hitomi
Kiyotaka
K
Shibata
Hideki
H
Maki
Masatoshi
M
D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D004815:Epidermal Growth Factor; D002154:Calpain; C117906:Capn7 protein, human