Analysis of xenon binding to photosystem II by X-ray crystallography.

In order to investigate oxygen binding and hydrophobic cavities in photosystem II (PSII), we have introduced xenon under pressure into crystals of PSII isolated from Thermosynechococcus elongatus and used X-ray anomalous diffraction analyses to identify the xenon sites in the complex. Under the conditions employed, 25 Xe-binding sites were identified in each monomer of the dimeric PSII complex. The majority of these were distributed within the membrane spanning portion of the complex with no obvious correlation with the previously proposed oxygen channels. One binding site was located close to the haem of cytochrome b559 in a position analogous to a Xe-binding site of myoglobin. The only Xe-binding site not associated with the intrinsic subunits of PSII was within the hydrophobic core of the PsbO protein.

Microtubule and motor-dependent endocytic vesicle sorting in vitro.

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process.

We have previously used the asialoglycoprotein receptor system to elucidate the pathway of hepatocytic processing of ligands such as asialoorosomucoid (ASOR). These studies suggested that endocytic vesicles bind to and travel along microtubules under the control of molecular motors such as cytoplasmic dynein. We now report reconstitution of this process in vitro with the use of a microscope assay to observe the interaction of early endocytic vesicles containing fluorescent ASOR with fluorescent microtubules. We find that ASOR-containing endosomes bind to microtubules and translocate along them in the presence of ATP. This represents the first time that mammalian endosomes containing a well-characterized ligand have been directly observed to translocate on microtubules in vitro. The endosome movement does not require cytosol or exogenous motor protein, is oscillatory, and is directed toward the plus and minus ends at equal frequencies. We also observe endosomes being stretched in opposite directions along microtubules, suggesting that microtubules could provide a mechanical basis for endocytic sorting events. The movement of endosomes in vitro is consistent with the hypothesis that microtubules actively participate in the sorting and distribution of endocytic contents.

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends.

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.

Physical and biological controls on carbon cycling in the equatorial pacific.

The equatorial Pacific is the largest oceanic source of carbon dioxide to the atmosphere and has been proposed to be a major site of organic carbon export to the deep sea. Study of the chemistry and biology of this area from 170 degrees to 95 degrees W suggests that variability of remote winds in the western Pacific and tropical instability waves are the major factors controlling chemical and biological variability. The reason is that most of the biological production is based on recycled nutrients; only a few of the nutrients transported to the surface by upwelling are taken up by photosynthesis. Biological cycling within the euphotic zone is efficient, and the export of carbon fixed by photosynthesis is small. The fluxes of carbon dioxide to the atmosphere and particulate organic carbon to the deep sea were about 0.3 gigatons per year, and the production of dissolved organic carbon was about three times as large. The data establish El Niño events as the main source of interannual variability.

Remote acoustic detection of a turbidity current surge.

A turbidity current surge has been detected in a leveed submarine channel in Rupert Inlet, British Columbia, with the use of acoustic sounders operating at 42.5, 107, and 200 kilohertz.

Oxygen consumption in pelagic marine sediments.

Measurements in the interstitial waters of pelagic red clay and carbonate ooze sediments in the central equatorial Pacific show that the dissolved oxygen content decreases with depth and levels off at nonzero values. The supply of reactive organic carbon introduced by bioturbation limits oxygen consumption at depth in the sediment. These gradients should produce diffusive fluxes across the sediment-water interface that average about 8.8 x 10(-14) mole per square centimeter per second or 0.08 milliliter per square meter per hour.