Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization,Interaction with the Clp Proteolytic Core,and Functional Importance

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.     

Polymix breeding with paternity analysis in Populus: a test for differential reproductive success (DRS) among pollen donors

Polymix breeding with paternity analysis (PMX/WPA) has been proposed as an alternative to traditional full-sib breeding and testing schemes. To fully capture the benefits of PMX/WPA, differential reproductive success (DRS) of pollen parents used in the polymix must be modest. DRS was evaluated in an operational test of PMX/WPA for a hybrid poplar breeding program. A 16-parent pollen polymix (Populus nigra L.) was used to pollinate seven clones of Populus deltoides (Bartr. ex. Marshall) under greenhouse breeding conditions. Progeny were grown out briefly and randomly sampled (357) prior to out-planting in field trials. Twenty-eight simple sequence repeat (SSR) loci were evaluated and 15 were selected for genetic characterization in small populations of three Populus spp (P. nigra, P. deltoides, and P. balsamifera spp trichocarpa Torr. & Gray). Seven loci were ultimately selected for paternity analysis of progeny. The average exclusion probability of the seven loci in P. nigra was 0.604; combined, the theoretical exclusion probability was 0.9999. However, only 95% of sampled progeny were unambiguously assigned a single paternal parent. Missing data likely accounted for most of the ambiguity. DRS was statistically significant though not prohibitive for practical utility of PMX/WPA as a breeding system. Of the 112 potential crosses in this study, 92 were represented. Eight of the 16 pollen parents contributed 83% of the progeny. Good pollen vigor, as measured by germination percent, did not ensure paternal success, but poor vigor was associated with lack of paternal success. PMX/WPA appears to be logistically and economically attractive for hybrid poplar breeding and testing.     

Nerve Growth Factor Amplifies Cyclic AMP Production in the HT4 Neuronal Cell Line

Abstract: There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml 15 min) amplified cAMP accumulation (≈75%) in response to activation of adenosine A₂ receptors (5 min) with 5′-N-ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down-regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF-induced amplification of cAMP accumulation. Although NGF-induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF-mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.     

Cross-linking of human multidrug resistance P-glycoprotein by the substrate, tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis. Evidence for rotation of a transmembrane helix.

We identified a thiol-reactive substrate, Tris-(2-maleimidoethyl)amine (TMEA), to explore the contribution of the TM segments 6 and 12 of the human multidrug resistance P-glycoprotein (P-gp) during transport. TMEA is a trifunctional maleimide and stimulated the ATPase activity of Cys-less P-gp about 7-fold. Cysteine-scanning mutagenesis of TM12 showed that the activity of mutant V982C was inhibited by TMEA. P-gp mutants containing V982C (TM12) and another cysteine in TM6 were constructed and tested for cross-linking with TMEA. A cross-linked product was observed in SDS-polyacrylamide gel electrophoresis for mutant L339C(TM6)/V982C(TM12). Cross-linking by TMEA also inhibited the ATPase activity of the mutant protein. Substrates such as cyclosporin A, vinblastine, colchicine, or verapamil inhibited cross-linking by TMEA. In the presence of ATP at 37 degrees C, cross-linking of mutant L339C/V982C was decreased. In contrast, there was enhanced cross-linking of mutant F343C(TM6)/V982C(TM12) in the presence of ATP. These results show that cross-linking must be within the drug-binding domain, that residues L339C(TM6)/V982C(TM12) must be at least 10 A apart, and that ATP hydrolysis promotes rotation of one or both TM helices.     

A reciprocal relationship between cutaneous nerves and repairing skin wounds in the developing chick embryo.

Various studies have suggested that the rate of adult skin healing may be in some way dependent on signals emanating from cutaneous nerves. Further, it appears that adult wounds become hyperinnervated by sensory nerves during the process of healing. In order to investigate this reciprocal relationship further, we have used a simple embryonic model to look at the effect of wounds on nerves, and conversely, the effect of nerves on wounds. We find that wounds made to the dorsum of the chick wing bud, at a stage prior to normal innervation (at E4), or soon after the normal establishment of cutaneous innervation (at E7), subtly alter the pattern of branching by perturbing developmental guidance cues, but do not cause hyperinnervation, whereas wounding at E14 does cause hyperinnervation. By creating chicks with nerveless wings, we show that from E7, wound healing in the absence of nerves is significantly impaired. These observations suggest that, from the earliest stages of skin innervation, the presence of nerves is beneficial to the healing process, but that, in contrast to neonatal and adult tissues, wound healing in the embryo and early foetus does not trigger hyperinnervation.     

The polypeptides of rat liver mitochondria: Identification of a 36,000 dalton polypeptide as the subunit of ornithine transcarbamylase

One of the major polypeptide bands seen after rat liver mitochondria are subjected to dodecyl sulfate gel electrophoresis is a component with a mass of 36,000 daltons that makes up 3 to 4% of the total mitochondrial protein. This band, designated Va (1), purifies with an enzyme of the urea cycle, ornithine transcarbamylase (E.C. 2.1.3.3). Evidence is presented that the band Va polypeptide is a single molecular species corresponding to the polypeptide chain of this enzyme.