Monica Lindén

Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical.

Hexoklnase of tumor cells [l] and certain normal tissues [2] are bound to the outer membrane of the mitochondrion. This binding is, at least in part, responsible for increased aerobic glycolysis, which characterizes rapidly growing tumor cells [3]. The rat liver outer membrane protein responsible for binding of hexokinase has been isolated and partially characterized [4]. The app. Mr reported for this peptide (31 000) is nearly identical to that of the outer membrane pore protein [5], purified and characterized in [6]. Since SDS electrophoresis of outer membranes reveals a single major band in the 3 1 000 Mr region, we have investigated the possibility that pore protein and the hexokinase-binding protein are identical. These results indicate that this is the case. [6,6’(n)-3H]Sucrose (l-5 Ci/mmol) was purchased from The Radiochemical Center (Amersham). [carboxyl-‘4C]Dextran (M, 70 000) with spec. act. 1 .l mCi/g was from New England Nuclear. Staphylococcus aureus V8 protease was purchased from Pharmacia (Uppsala). Chymotrypsin, soy bean lecithin and octyl-fl-D-glucopyranoside were all purchased from Sigma. Ampholines were from LKB (Stockholm).

Effects of Desmin Gene Knockout on Mice Heart Mitochondria

In heart tissue from mice lacking the intermediate filament (IF) desmin, mitochondria show an abnormal shape and distribution (Thornell et al., 1997). In the present study we have isolated heart mitochondria from desmin null (D−/−) and control (D+/+) mice, and analyzed their composition by SDS–PAGE, immunoblotting, and enzyme measurements. We found both in vitro and in situ that the conventional kinesin, the microtubule-associated plus-end directed motor, was frequently associated with D+/+ heart mitochondria, but not with D−/− heart mitochondria, suggesting that the positioning of mitochondria in heart is a dynamic event involving the IF desmin, the molecular motor kinesin, and, most likely, the microtubules (MT) network. Furthermore, an increased capacity in energy production was found, as indicated by a threefold higher creatine kinase activity in heart mitochondria from D−/− compared to D+/+ mice. We also observed a significantly lower amount of cytochrome c in heart mitochondria from D−/− mice, and a relocalization of Bcl-2, which may indicate an apoptotic condition in the cell leading to the earlier reported pathological events, such as cardiomyocytes degeneration and calcinosis of the heart (Thornell et al., 1997).

Hydrodynamic properties of porin isolated from outer membranes of rat liver mitochondria.

The hydrodynamic properties of purified porin (Mr = 30 000), isolated from outer membranes of rat liver mitochondria has been studied. After gel filtration, active porin was eluted in a symmetrical peak with an estimated Stokes radius of 5.4 nm. The sedimentation coefficient (s) and partial specific volume (v) were found to be 2.6 S and 0.908 cm3/g, respectively, for the purified porin-Triton X-100 complex. Based on these determinations, a molecular weight of 170 000 for the porin-Triton X-100 complex was calculated. Correcting for bound Triton X-100, 1.8 g/g of protein, a molecular weight of 60 000 was estimated for the protein portion of the complex. Thus, isolated active porin appears to exist as a dimer.

Subfractionation of the outer membrane of rat brain mitochondria: evidence for the existence of a domain containing the porin-hexokinase complex

Isolated and well-characterized rat brain nonsynaptic mitochondria were subfractionated by digitonin. Antibodies to a uniquely outer membrane protein, porin, have allowed us to use this protein for the first time as an outer membrane marker in brain. Hexokinase, which binds to porin, was also measured. Based upon the sequential release of these and other marker enzymes with increasing concentrations of digitonin, three outer membrane domains have been identified. Two populations of porin were found by this treatment. The most plausible interpretation of our results is that the two porin populations exist in different membrane environments with regard to cholesterol. One of these populations binds most of the hexokinase and appears to be associated with the inner membrane. It is proposed that the porin-hexokinase complex in brain mitochondria is located in a cholesterol-free membrane domain together with inner membrane components. This domain has the features of contact points which have been visualized by electron microscopy.

Studies on the interaction between mitochondria and the cytoskeleton

Mitochondrial movements and morphology are regulated through interactions with the cytoskeletal system, in particular the microtubules. An interaction between the microtubule-associated proteins (MAPs) and the outer surface of rat brain mitochondria has been demonstratedin vitro andin situ. One of the MAPs, MAP2, binds to specific high-affinity sites on the outer membrane. Upon binding, MAP2 is released from microtubules, and it induces a physical alteration in the outer membrane which is characterized by a tighter association of porin with the membrane. It is concluded that MAP2 either binds to porin or to a domain of the outer membrane which alters the membrane environment of porin. The possibility is raised that this domain participates in mitochondrial mobilityin situ.

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.

Studies on the relationship between the inner and outer membranes of rat liver mitochondria as determined by subfractionation with digitonin.

The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria.

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.

In vitro studies of the physical interactions between neurofilaments, microtubules and mitochondria isolated from the central nervous system

In order to explore the molecular nature and the regulation of dense cytomatrix which interconnects MT, NF and membranous organelles in neurons (9), the interactions between NF, MT and each of these cytoskelatal elements with brain mitochondria were investigated in vitro using biochemical and viophysical methods. From these studies, the following conclusions were drawn: 1‐ Pure NF form in vitro a highly viscous gel, dependent upon the phosphorylation state of the side arms of the NF‐H and M subunits which might participate directly to the interactions since antibodies specific of these phosphorylated sites inhibited efficiently the NF gelation. This process is modulated by both ATP hydrolysis and soluble molecules from nervous tissue and it might reflect the highly controled organization of NF bundles in axons. 2‐ In contrast with NF, low viscosity levels were detected in MT suspensions. However, the occurrence of weak interactions between MT were deduced from studies with taxol, ATP, AMP‐PNP and Mg ions,...

Biogenesis of the outer mitochondrial membrane in isolated rat hepatocytes

The outer membrane from rat liver mitochondria was first isolated in 1966 [ 1,2]. Since then very little attention has been paid to the synthesis and assembly of the outer membrane. Today synthesis and assembly of the outer membrane is becoming increasingly important, particularly in view of its possible role in import of cytoplasmically made matrix proteins [3]. For example, to explain the specific recognition of imported proteins by the mitochondrion, several investigators have proposed the presence of receptor proteins in the outer membrane [3-S]. If such a receptor exists, however, one must solve the paradox as to how the receptor protein recognizes the mitochondrial membrane. One obvious possibility is that such protein might be coded for by mitochondrial DNA. This is also suggested by earlier experiments, which showed a small but significant labeling of the outer membrane in in vitro labeled mitochondria [6,7]. Here, we have studied biosynthesis of the outer mitochondrial membrane in isolated rat hepatocytes. Our results show that all of the peptides of the outer membrane, including the pore protein [8], are translated on cytoplasmic ribosomes.