Adenine nucleotide transport in plants: much more than a mitochondrial issue

Adenine nucleotides play a vital role in plant metabolism and physiology, essentially representing the major energy currency of the cell. Heterotrophic cells regenerate most of the ATP in mitochondria, whereas autotrophic cells also possess chloroplasts, representing a second powerhouse for ATP regeneration. Even though the synthesis of these nucleotides is restricted to a few locations, their use is nearly ubiquitous across the cell and thereby highly efficient systems are required to transport these molecules into and out of different compartments. Here, we discuss the location, biochemical characterization and evolution of corresponding transport systems in plants. We include recent scientific findings concerning organellar transporters from plants and algae and also focus on the physiological importance of adenine nucleotide exchange in these cells.     

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.     

The [FeFe]-hydrogenase maturation protein HydF contains a H-cluster like [4Fe4S]-2Fe site

Formation of the catalytic six-iron complex (H-cluster) of [FeFe]-hydrogenase (HydA) requires its interaction with a specific maturation protein, HydF. Comparison by X-ray absorption spectroscopy at the Fe K-edge of HydF from Clostridium acetobutylicum and HydA1 from Chlamydomonas reinhardtii revealed that the overall structure of the iron site in both proteins is highly similar, comprising a [4Fe4S] cluster (Fe–Fe distances of ∼2.7 Å) and a di-iron unit (Fe–Fe distance of ∼2.5 Å). Thus, a precursor of the whole H-cluster is assembled on HydF. Formation of the core structures of both the 4Fe and 2Fe units may require only the housekeeping [FeS] cluster assembly machinery of the cell. Presumably, only the 2Fe cluster is transferred from HydF to HydA1, thereby forming the active site.     

Protein kinase D increases maximal Ca2+-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser315

Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.     

Late stage infection in sleeping sickness

At the turn of the 19(th) century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.     

Role of the 1,25D3-MARRS receptor in the 1,25(OH)2D3-stimulated uptake of calcium and phosphate in intestinal cells

We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.     

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.