Novel Mitochondrial Creatine Transport Activity

Abstract

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N-and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of ϳ70 and ϳ55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [ 14 C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentrationdependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

The Nucleoside Diphosphate Kinase D (NM23-H4) Binds the Inner Mitochondrial Membrane with High Affinity to Cardiolipin and Couples Nucleotide Transfer with Respiration

“…Isolation of Purified Mitochondria and of Submitochondrial Fractions-Mitochondria from male Wistar rat liver were isolated by differential centrifugation and Percoll gradient according to Hovius et al (29) with some modifications (30). Importantly, swelling of the pure mitochondrial preparation was carried out in buffer containing 30% glycerol, which disrupts the mitochondrial contact sites between mitochondrial inner membrane and outer membrane.…”

Controlling the Flow of Energy: Inhibition and Stimulation of the Creatine Transporter

“…Both immunoflurescence and immunoelectron microscopy studies demonstrated two major creatine transporter protein species of ~55 and ~70kDa associated with the inner mitochondrial membrane, as would be expected for a mitochondrial transporter, both having a low affinity (high K m~1 5mM ) to creatine [3]. Western blot studies indicated that a creatine transporter having molecular mass ~58kDa is located in the plasma membrane and is responsible for high affinity (low K m ~ 20-50 μM - [59]) uptake of creatine [59].…”

“…These data suggest that creatine transporter is a system made up of two separate creatine transporters: the high affinity plasma membrane creatine transporter (PM-CRT) and the low affinity mitochondrial creatine transporter (Mi-CRT). They suggest a compartmentation of two intracellular creatine pools, in the cytosol and in mitochondria, respectively, in addition to a third pool, blood creatine [3].…”

“…Studies on creatine transporter expression showed the presence of two different gene products corresponding to two major polypeptides of ~55 and ~70kDa on Western blots [3,[55][56][57][58], that are thought to be generated by alternative splicing [55], as well as a creatine transport isoform with an apparent molecular mass of ~58kDa [59]. In ion-dependency experiments carried out in oocites it was shown that CRT has a stoichiometry of 2Na + and 1 Cl - [60] and studies on the uptake of radioactive creatine or other guanidino compounds showed that a carboxyl group and a guanidino group, separated by 2-3 carbon atoms, are important for substrate recognition by creatine transport, whereas the N-methyl group seems not to be critical.…”

“…In fact even in vitro, where there is no blood-brain barrier, the pharmacological inhibition of the transporter prevents creatine uptake by mouse hippocampal slices [14,15]. Transporter-mediated transfer of creatine from the extracellular to the intracellular space is mono-directional and occurs against a very high concentration gradient (600-1000 fold) that is due to a high creatine content in neurons and glial cells [3]. Despite the presence of the transporter, crossing of the blood-brain barrier by creatine is slow and limited [16,17].…”