Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

Abstract

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KC1 and 20 mM EDTA, and subjected to shearing. Excluding KCI, EDTA, or shearing from the procedure resulted in incomplete separation.Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way from animals injected with [asS]methionine _ cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

“…The existence of anchorage sites between ER and mitochondria have been suggested based on co-sedimentation of ER particles with mitochondria and electron microscopic observations of close associations between mitochondria and ER vesicles [151,152]. Recent studies of the ER-mitochondrial interface by electron tomography and by limited proteolysis provided evidence for direct protein linkage (tethers) between both rough and smooth ER and mitochondria.…”

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

“…MCSs have been observed by both biochemical methods and electron microscopy in mammalian, yeast, fly, and plant cells (Shore and Tata 1977;Perkins et al 1997;Ladinsky et al 1999). These dynamic structures are highly enriched in lipid biosynthetic enzymes and LTPs, and are generally formed and/or stabilized by specialized tethering proteins (Fig.…”

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

“…Structural interactions between SR/ER and mitochondria in different cell types A close association between ER and mitochondria was initially described in electron microscopy images of liver tissue (Morré et al, 1971;Shore and Tata, 1977). A stable interorganellar interaction was indicated when tissue fractionation studies revealed an ER membrane contamination in isolated mitochondria, referred to as mitochondria-associated membranes (MAMs) (Vance, 1990).…”