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J. Biol. Chem., Vol. 278, Issue 18, 16336-16346, May 2, 2003
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From the Ubiquitously expressed calpains are
Ca2+-dependent, intracellular cysteine
proteases comprising a large catalytic subunit (domains DI-DIV) and a
noncovalently bound small regulatory subunit (domains DV and DVI). It
is unclear whether Ca2+-induced calpain activation is
followed by subunit dissociation or not. Here, we have applied advanced
fluorescence microscopy techniques to study calpain subunit
interactions in living cells using recombinant calpain subunits or
domains fused to enhanced cyan and enhanced yellow fluorescent reporter
proteins. All of the overexpressed variants of the catalytic
subunit (DI-IV, DI-III, and DI-IIb) were active and
Ca2+-dependent. The intact large subunit, but
not its truncated variants, associates with the small subunit under
resting and ionomycin-activated conditions. All of the variants were
localized in cytoplasm and nuclei, except DI-IIb, which accumulates in
the nucleus and in nucleoli as shown by microscopy and cell
fractionation. Localization studies with mutated and chimeric variants
indicate that nuclear targeting of the DI-IIb variant is conferred by
the two N-terminal helices of DI. Only those variants that contain DIII
migrated to membranes upon the addition of ionomycin, suggesting that
DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain,
but not dissociation of its intact subunits, contribute to regulate its
proteolytic activity in vivo.
Subcellular Localization and in Vivo
Subunit Interactions of Ubiquitous µ-Calpain*
§¶,,
,,,,,§§,,
Abteilung für Klinische Chemie und
Klinische Biochemie, Chirurgische Klinik Innenstadt, Klinikum der
Ludwig-Maximilians-Universität, D-80336 München,
§ Adolf-Butenandt-Institut der
Ludwig-Maximilians-Universität, D-80336 München,
Deutsches Krebsforschungszentrum, D-69120 Heidelberg,
** Physiologisches Institut der
Ludwig-Maximilians-Universität, D-80336 München,
Institut für Molekularbiologie und
Zellkulturtechnik, Fachhochschule Mannheim, D-68163 Mannheim, and
¶¶ Max-Planck-Institut für Biochemie,
D-82152 Martinsried, Germany
*
This work was supported by Grants A3 (to E. A. A.) and A6 (to W. M.) from the Sonderforschungsbereich 469 of the Ludwig-MaximiliansUniversität München.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.
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