Journal
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
Volume 306, Issue 5, Pages H764-H770Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpheart.00909.2013
Keywords
matrix metalloproteinase 2; mitochondria-associated membrane; mitochondria; calreticulin
Funding
- Canadian Institutes of Health Research Grant [MOP66953]
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Matrix metalloproteinase-2 (MMP-2) has been extensively studied in the context of extracellular matrix remodeling but is also localized within cells and can be activated by prooxidants to proteolyze specific intercellular targets. Although there are reports of MMP-2 in mitochondria, a critical source of cellular oxidative stress, these studies did not take into account the presence within their preparations of the mitochondria-associated membrane (MAM), a subdomain of the endoplasmic reticulum (ER). We hypothesized that MMP-2 is situated in the MAM and therefore investigated its subcellular distribution between mitochondria and the MAM. Immunogold electron microscopy revealed MMP-2 localized in mitochondria of heart sections from mice. In contrast, immunofluorescence analysis of an MMP-2: HaloTag fusion protein expressed in HL-1 cardiomyocytes showed an ER-like distribution, with greater colocalization with an ER marker (protein disulfide isomerase) relative to the mitochondrial marker, MitoTracker red. Although MMP-2 protein and enzymatic activity were present in crude mitochondrial fractions, once these were separated into purified mitochondria and MAM, MMP-2 was principally associated with the latter. Thus, although mitochondria may contain minimal levels of MMP-2, the majority of MMP-2 previously identified as mitochondrial is in fact associated with the MAM. We also found that calreticulin, an ER-and MAM-resident Ca2+ handling protein and chaperone, could be proteolyzed by MMP-2 in vitro. MAM-localized MMP-2 could therefore potentially impact mitochondrial function by affecting ER-mitochondrial Ca2+ signaling via its proteolysis of calreticulin.
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