Bacterial rhodopsin: evidence for a new type of phototrophy in the sea.

Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps. So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea. We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton.

Proton transport by sensory rhodopsins and its modulation by transducer-binding.

The study of light-induced proton transfers in the archaeal sensory rhodopsins (SR), phototaxis receptors in Halobacterium salinarum, has contributed important insights into their mechanism of signaling to their cognate transducer subunits in the signaling complex. Essential features of the bacteriorhodopsin (BR) pumping mechanism have been conserved in the evolution of the sensors, which carry out light-driven electrogenic proton transport when their transducers are removed. The interaction of SRI with its transducer blocks proton-conducting channels in the receptor thereby inhibiting its proton pumping, indicating that the pump machinery, rather than the transport activity itself, is functionally important for signaling.

Variable surface loops and myosin activity: accessories to a motor.

The catalytic head of myosin is a globular structure that has historically been divided into three segments of 25, 50, and 20 kDa. The solvent-exposed, proteolytically-sensitive surface loops of myosin that join these three segments are highly variable in their sequences. While surface loops have not traditionally been thought to affect enzymatic activities, these loops lie near the ATP and actin-binding sites and have been implicated in the modulation of myosin's kinetic activities.

Dynacortin, a genetic link between equatorial contractility and global shape control discovered by library complementation of a Dictyostelium discoideum cytokinesis mutant.

We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect.

Myosin-V stepping kinetics: a molecular model for processivity.

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step.

Color-sensitive motility and methanol release responses in Rhodobacter sphaeroides.

Blue-light-induced repellent and demethylation responses, characteristic of behavioral adaptation, were observed in Rhodobacter sphaeroides. They were analyzed by computer-assisted motion analysis and through the release of volatile tritiated compounds from [methyl-(3)H]methionine-labeled cells, respectively. Increases in the stop frequency and the rate of methanol release were induced by exposure of cells to repellent light signals, such as an increase in blue- and a decrease in infrared-light intensity.

Towards a molecular understanding of cytokinesis.

In this review, we focus on recent discoveries regarding the molecular basis of cleavage furrow positioning and contractile ring assembly and contraction during cytokinesis. However, some of these mechanisms might have different degrees of importance in different organisms. This synthesis attempts to uncover common themes and to reveal potential relationships that might contribute to the biochemical and mechanical aspects of cytokinesis.

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor.

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540.

Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change.

The dynamic assembly/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization. Phosphorylation of three threonines, 1823, 1833 and 2029, in the tail of Dictyostelium discoideum myosin II has been implicated in control of myosin filament assembly. By systematically replacing the three threonines to aspartates, mimicking a phosphorylated residue, we found that position 1823 is the most critical one for the regulation of myosin filament formation and in vivo function.

A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II-null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II-null cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control.

A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment.

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture.

Transducer-binding and transducer-mutations modulate photoactive-site-deprotonation in sensory rhodopsin I.

Sensory rhodopsin I (SRI) is a seven-transmembrane helix retinylidene protein that mediates color-sensitive phototaxis responses through its bound transducer HtrI in the archaeon Halobacterium salinarum. Deprotonation of the Schiff base attachment site of the chromophore accompanies formation of the SRI signaling state, S(373). We measured the rate of laser flash-induced S(373) formation in the presence and absence of HtrI, and the effects of mutations in SRI or HtrI on the kinetics of this process.

Proton circulation during the photocycle of sensory rhodopsin II.

Sensory rhodopsin II (SRII) in Halobacterium salinarum membranes is a phototaxis receptor that signals through its bound transducer HtrII for avoidance of blue-green light. In the present study we investigated the proton movements during the photocycle of SRII in the HtrII-free and HtrII-complexed form. We monitored sustained light-induced pH changes with a pH electrode, and laser flash-induced pH changes with the pH indicator pyranine using sealed membrane vesicles and open sheets containing the free or the complexed receptor.

Identification of methylation sites and effects of phototaxis stimuli on transducer methylation in Halobacterium salinarum.

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography.

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin.

Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum.

An expansion accompanying the formation of the first intermediate in the photocycle of transducer-free sensory rhodopsin I (SRI) was determined by means of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is approximately 5.5 mL and for the mutant SRI-D76N, approximately 1.

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C.

Specialized conservation of surface loops of myosin: evidence that loops are involved in determining functional characteristics.

The molecular motor myosin has been the focus of considerable structure-function analysis. Of key interest are the portions of the protein that control the rate of ATP hydrolysis, the affinity for actin, and the velocity at which myosin moves actin. Two regions that have been implicated in determining these parameters are the "loop" regions at the junctions of the 25 kDa and 50 kDa domains and the 50 kDa and 20 kDa domains of the protein.

Single-molecule biomechanics with optical methods.

Single-molecule observation and manipulation have come of age. With the advent of optical tweezers and other methods for probing and imaging single molecules, investigators have circumvented the model-dependent extrapolation from ensemble assays that has been the hallmark of classical biochemistry and biophysics. In recent years, there have been important advances in the understanding of how motor proteins work.

The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices.

Chimeras of the Halobacterium salinarum transducers HtrI and HtrII were constructed to study the structural determinants for their specific interaction with the phototaxis receptors sensory rhodopsins I and II (SRI and SRII), respectively. Interaction of receptors and transducers was assessed by two criteria: phototaxis responses by the cells and transducer-modulation of receptor photochemical reaction kinetics in membranes. Coexpression of HtrI with SRII or HtrII with SRI did not result in interaction by either criterion.

Myosin II localization during cytokinesis occurs by a mechanism that does not require its motor domain.

Myosin II generates force for the division of eukaryotic cells. The molecular basis of the spatial and temporal localization of myosin II to the cleavage furrow is unknown, although models often imply that interaction between myosin II and actin filaments is essential. We examined the localization of a chimeric protein that consists of the green fluorescent protein fused to the N terminus of truncated myosin II heavy chain in Dictyostelium cells.

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway.

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism.

Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II.

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin-myosin-ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket.