The physical association between rat liver mitochondria and rough endoplasmic reticulum. I. Isolation, electron microscopic examination and sedimentation equilibrium centrifugation analyses of rough endoplasmic reticulum-mitochondrial complexes
Pickett, C.B.; Montisano, D.; Eisner, D.; Cascarano, J.
Experimental Cell Research 128(2): 343-352
Rough endoplasmic reticulum-mitochondrial (RER-MT) complexes were isolated from rat liver homogenates by rate zonal centrifugation using a reorienting zonal rotor. EM examination of the isolated complexes reveals a close association between rough endoplasmic reticulum (RER) and mitochondria. The associated RER appears as bilamellar sheets as it does in intact liver tissue, not as microsomal vesicles. When the complexes are subjected to sedimentation equilibrium centrifugation, the marker enzymes for mitochondria and RER coband at an equilibrium density of 1.190. EM analysis of the complexes after sedimentation equilibrium centrifugation again reveals a close association between RER and mitochondria. Treatment of the complexes with 500 mM KCl or 500 mM KCl plus 20 mM EDTA resulted in a shift in the equilibrium density of the complexes to 1.180 and 1.176, respectively. Concomitant with the density shift was a release of A260 units to the top of the gradient. After incubating KCl-EDTA stripped complexes with cytoplasmic ribosomes and ribosomal subunits, the complexes band at the same equilibrium density, 1.190, as do untreated complexes. To completely remove the associated RER it is necessary to treat the complexes with digitonin at a concentration of 0.13 mg digitonin/mg protein. A fraction of the total cellular RER is probably physically associated with rat liver mitochondria.