Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.
Ubiquitously expressed calpains are Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
Gil-Parrado
Shirley
S
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik Innenstadt, Klinikum der Ludwig-Maximilians-Universität, Nussbaumstrasse 20, D-80336 Münich, Germany. shirgalpa@web.de
Popp
Oliver
O
Knoch
Tobias A
TA
Zahler
Stefan
S
Bestvater
Felix
F
Felgenträger
Marcel
M
Holloschi
Andreas
A
Fernández-Montalván
Amaury
A
Auerswald
Ennes A
EA
Fritz
Hans
H
Fuentes-Prior
Pablo
P
Machleidt
Werner
W
Spiess
Eberhard
E
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis; D002154:Calpain / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D002135:Calcium-Binding Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C033668:calpastatin; D002154:Calpain; C449002:mu-calpain; D002118:Calcium