Quasi-synaptic calcium signal transmission between endoplasmic reticulum and mitochondria.

Transmission of cytosolic [Ca2+] ([Ca2+]c) oscillations into the mitochondrial matrix is thought to be supported by local calcium control between IP3 receptor Ca2+ channels (IP3R) and mitochondria, but study of the coupling mechanisms has been difficult. We established a permeabilized cell model in which the Ca2+ coupling between endoplasmic reticulum (ER) and mitochondria is retained, and mitochondrial [Ca2+] ([Ca2+]m) can be monitored by fluorescence imaging. We demonstrate that maximal activation of mitochondrial Ca2+ uptake is evoked by IP3-induced perimitochondrial [Ca2+] elevations, which appear to reach values >20-fold higher than the global increases of [Ca2+]c. Incremental doses of IP3 elicited [Ca2+]m elevations that followed the quantal pattern of Ca2+ mobilization, even at the level of individual mitochondria. In contrast, gradual increases of IP3 evoked relatively small [Ca2+]m responses despite eliciting similar [Ca2+]c increases. We conclude that each mitochondrial Ca2+ uptake site faces multiple IP3R, a concurrent activation of which is required for optimal activation of mitochondrial Ca2+ uptake. This architecture explains why calcium oscillations evoked by synchronized periodic activation of IP3R are particularly effective in establishing dynamic control over mitochondrial metabolism. Furthermore, our data reveal fundamental functional similarities between ER-mitochondrial Ca2+ coupling and synaptic transmission.     

Connections between mitochondria and endoplasmic reticulum in rat liver and onion stem

Summary Smooth-surfaced elements of endoplasmic reticulum contact and are attached to the outer membranes of mitochondria in rat liver and onion stem. Some connections appear as short, 150–300 Å diameter tubules that bridge the space between the conjoining elements. In liver, the smooth-surfaced endoplasmic reticulum cisternae connected to the outer mitochondrial membrane are shown to be continuous with rough-surfaced endoplasmic reticulum. Here, the smooth-surfaced endoplasmic reticulum is identified in negatively stained preparations of isolated cell fractions and in thin sections of tissues by the presence of lipoprotein particles characteristic of this cell component. In onion, the identification of endoplasmic reticulum is based on continuity with rough-surfaced endoplasmic reticulum.     

Transfer of phospholipids between the endoplasmic reticulum and mitochondria in rat hepatocytes in vivo

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling $\text{in}\text{vivo}$. With [³H]glycerol microsomal phosphatidylethanolamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occurred only after about 5 hours. It appears that the $\text{in}\text{vivo}$ transfer of these phospholipids between the two membrane compartments is a relatively slow process.     

Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca(2+) concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 microM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.     

Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.     

Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail‐anchored proteins

Tail‐anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein‐free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5‐ER and b5‐RR, which differ only in the charge of the C‐terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell‐free and in cellula results, we analyzed targeting in digitonin‐permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C‐terminal negative charge of b5‐ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ES_I) reduced insertion of b5‐ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ES_I, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C‐terminal charge, and reveal a novel, substrate‐specific ER‐targeting pathway.      

Dithiothreitol and the translocation of preprolactin across mammalian endoplasmic reticulum

The translocation mode of preprolactin (pPL) across mammalian endoplasmic reticulum was reinvestigated in light of recent findings that nascent secretory polypeptides synthesized in the presence of a highly reducing environment could be translocated posttranslationally and independently of their attachment to the ribosome (Maher, P. A., and S. J. Singer, 1986, Proc. Natl. Acad. Sci. USA, 83:9001-9005). The effects of the reducing agent dithiothreitol (DTT) on pPL synthesis and translocation were studied in this respect. The translocation of pPL was shown to take place only cotranslationally. The apparent posttranslational translocation was due to ongoing chain synthesis irrespective of the presence of high concentrations of DTT. When synthesis was completely blocked, no translocation was observed in the presence or absence of DTT. The synthesis of pPL was retarded by DTT, while its percent translocation was enhanced. The retardation in synthesis was reflected in reduced rates of initiation and elongation. As a consequence of this retardation, which increases the ratio of microsomes to nascent chains, and of possible effects on the conformation of nascent pPL and components of the translocation apparatus, DTT may expand the time and chain length windows for nascent chain translocation competence.     

Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

Regulatory interaction between the respiratory chain of mitochondria and the oxidative system of the endoplasmic reticulum]

A regulation mechanism of the interaction of microsomal oxidases and mitochondrial respiratory chain with oxidase substrates is suggested. Quantitative comparison of their affinity to microsomal oxidases and mitochondrial NADH-dehydrogenase is carried out. The interaction with both systems is found to be hydrophobic. It is found that microsomal oxidase substrates inhibit mitochondrial NADH-dehydrogenase at concentrations, which should completely fill the active site of cytochrome P-450. It is suggested that redistribution of reduced equivalents from mitochondria to microsomes and the acceleration of xenobiotic detoxication take place.