Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle     

Kinetics of channelized membrane ions in magnetic fields

The cyclotron resonance model for channel ion transport in weak magnetic fields is extended to include damping losses. The conductivity tensor is obtained for different electric field configurations, including the circuital field E phi normal to the channel axis. The conductivity behavior close to the cyclotron resonance frequency omega c is compared to existing Ca2+-efflux data in the literature. A collision time of .023 s results from this comparison under the assumption that K+ ions are transiting in a 0.35 G field. We estimate a mean kinetic energy of 3.5 eV for this ion at resonance. This model leads to discrete modes of vibration (eigenfrequencies) in the ion-lattice interaction, such that omega n = n omega c. The presence of such harmonics is compatible with recent results by Blackman et al. [1985b] and McLeod et al. [1986] with the interesting exception that even modes do not appear in their observations, whereas the present model has no restriction on n. This harmonic formalism is also consistent with another reported phenomenon, that of quantized multiple conductances in single patch-clamped channels.     

From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility

Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Water transport parameters and regulatory processes inEremosphaera viridis

Summary The water relations parameters and the osmoregulatory response ofEremosphaera viridis were investigated both by using the pressure probe technique and by analyzing the intracellular pool of osmotically active agents. In the presence of various concentrations of different salts a biphasic osmoregulatory response was recorded, consisting of a rapid decrease in turgor pressure due to water loss followed by an increase in turgor pressure to the original turgor pressure value (depending on the salt). The values of turgor pressure, volumetric elastic modulus and hydraulic conductivity depended on the composition of the media. Nonelectrolytes did not cause a turgor recovery after the initial water efflux. The second phase of turgor regulation in the presence of salts was characterised by the intracellular accumulation of ions and sugars and required at least 24 hr. Analysis of the cell sap showed that the increase in the internal osmotic pressure was mainly achieved by accumulation of sucrose. Additionally, accumulation of glucose was observed in illuminated cells in the presence of Rb and K. Electron micrographs suggested that the sucrose was produced by degradation of starch granules. Turgor pressure recovery after salt stress seemed to be dependent on temperature and is well correlated with the according photosynthetic activity. The data suggest that a temperature-dependent enzyme which is activated by potassium or rubidium is involved in the regulatory response.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.